Genetics&Genomics

Dna Extraction Promega Dnaiq System Database Protocol

Rooted hair samples

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Always??wear gloves??and use??barrier (filter) tips??when performing this protocol. To prevent confusion and contamination, never process the hair from more than 4 donors at one time.

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Preparation:

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Before beginning, put down a fresh piece of bench paper over the area in which you will be working and then make sure you have all of the following on the work table:

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  • Your personal DNA extraction kit
  • Clean microscope slides and cover slips
  • Several sterile scalpels
  • Ice bucket with ice (or freezer tube holder)
  • Box of gloves (your size)
  • A box of filter tips (small size)
  • Plastic tube rack
  • 1 tube of 10 mg/mL proteinase K from your personal freezer box (placed in tube rack to thaw)
  • 1 tube 1 M DTT from your freezer box (placed in tube rack to thaw)
  • 1 tube Incubation Buffer from your freezer box (placed in tube rack to thaw)
  • No more than 4 CSI-TRU hair tubes (taken from the freezer)
  • Pair of tweezers
  • Container of sterile 1.5 mL microcentrifuge tubes
  • DNA-IQ magnetic stand
  • 10% bleach solution
  • Biohazard waste container
  • Set of micropipettors
  • Calculator
  • Timer
  • Ultra-fine point sharpie marker
  • Lab notebook
  • Gel pen for writing in lab notebook

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Also check to make sure that following equipment is available and in working order:

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  • Water bath set at 56 degrees C
  • Tube heater set at 65 degrees C with 1.5-mL tube block in place
  • Microcentrifuge
  • Vortexer
  • Microscope

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Procedure:

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1.???????????? Turn on the water bath and tube heater (if not already on).

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2.???????????? Remove your donor tubes containing hair from the freezer and place them on the ???????? bench near the microscope.

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3.???????????? Open the first donor tube and examine each hair under the microscope, placing ?????????????? the root-end of the hair on a clean microscope slide and covering it with a cover slip ?????? to prevent movement during examination. If you can’t tell which end has the root, ???????? examine both ends under the microscope until you can determine the hair’s orientation.

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4.???????????? Cut the end of each hair about 1 cm from the root using a sterile scalpel. Then use ???? your tweezers to transfer the root ends into a clean, labeled 1.5-mL microcentrifuge ?????????? tube.

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????????????????????Although you will examine and cut all the hairs in each donor’s tube, retain ???????????? half the roots in case you need them later. These ends should be ???????????? placed in a sterile 1.5-mL microcentrifuge tube that is clearly labeled with ???????????????????? the TRU-Sample # and returned to the collection bag for that donor (in the ???????????????????? freezer).

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5.???????????? Calculate the total amount of incubation/DTT/proteinase K buffer you will need ?????????????????? according to the following formula (per sample):

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???????????????????????????????????????????????????????? Incubation buffer???????????????????????????? ?? ????80 uL

???????????????????????????????????????????????????????? 1 M DTT???????????????????????????????????????????????????? ?? ????10 uL

???????????????????????????????????????????????????????? proteinase K solution???????????????????? ?? ????10 uL

???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ???? 100 uL

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?????????????????? For example, if you are planning to process the hair from four donors, you would ???????? need to make up 400 uL of this buffer.

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6.???????????? Make up the incubation/DTT/proteinase K buffer and gently mix it by inverting several times.??Then store it on ice or in a freezer tube holder throughout ???????????????????? the remainder of the procedure.

7.???????????? Add 100 uL incubation/DTT/proteinase K buffer to each of the 1.5-mL tubes ?????? containing the donor hair roots. Invert several times to mix and then incubate the tubes in the 56 degree water bath for 1 hour.Make sure that the hairs are ?????????? completely covered by the buffer during this incubation. You can use a ?????????????????????? sterile toothpick to push the hairs under the buffer solution, if needed.

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8.???????????? While you are waiting, calculate the total amount of lysis buffer you will need to ?????????????? prepare by multiplying the number of hair donors by 300 uL. (For 4 hair donor tubes, ?????????? 1,200 uL of lysis buffer would be needed.)

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9.???????????? Invert the tube of lysis buffer (DNA extraction kit) several times to mix and then ?????????? aliquot the calculated volume into a sterile 1.5-mL microcentrifuge tube.

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10.???????? Mix the 1 M DTT by vortexing for a few seconds. Then add 1 uL of 1 M DTT for every ???????????????? 100 uL of lysis buffer.

11.???????? Mix the lysis buffer and DTT together by inverting the tube several times.

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12.???????? When the incubation of the tubes is complete, remove them from the water bath???????????? and add 200 uL of lysis buffer/DTT mixture and 7 uL of DNA-IQ resin to each tube. ???????????????? (Make sure to vortex the resin to ensure that it is well-mixed before aliquoting.)

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13.???????? Vortex the tubes at high speed for 3 seconds and then incubate at room temperature ?????????????????? for 5 minutes.

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14.???????? Vortex the tubes at high speed. The place then in the magnetic stand. Separation of ?? the resin from the surrounding solution should occur instantly.??If the resin does ?????? not form a distinct pellet on the side of the tube, vortex the tube and quickly ???????????????????? place it back in the stand.

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15.???????? Using a micropipettor, carefully remove and discard the solution from each tube, ?????????? being careful not to disturb the resin pellet (which is now bound to the DNA).

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16.???????? Add 100 uL of lysis/DTT buffer to each tube. Remove the tubes from the magnetic ?????? stand and vortex for 2 seconds at high speed.

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17.???????? Return the tubes to the magnetic stand. Carefully remove and discard all of the lysis buffer, being careful not to disturb the resin pellet.

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18.???????? Add 100 uL Wash Buffer to each tube. Remove the tubes from the magnetic stand ?????? and vortex for 2 seconds at high speed.

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19.???????? Return the tubes to the magnetic stand and remove and discard all of the Wash ?????????????? Buffer.

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20.???????? Repeat steps 18-19 two more times for a total of 3 washes. Make sure that all of the ?????????????????? solution has been removed from each tube after the last wash.

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21.???????? With the tubes in the magnetic stand the lids open, air-dry the resin for 5 minutes. ????????Do not dry the resin for more than 20 minutes, as this may inhibit the ?????????????? removal of DNA.

22.???????? Add 50 uL of elution buffer (DNA extraction kit) to each tube.

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23.???????? Close the lids and vortex for 2 seconds at high speed. Then incubate the tubes in the 65 degree tube heater for 5 minutes.

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24.???????? Remove the tubes from the heater and vortex for 2 seconds at high speed. Then ?????????? immediately place the tubes in the magnetic stand.??The tubes must remain hot as ?????????????????? they are placed in the magnetic stand, or yield will decrease.

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25.???????? Using a micropipettor, carefully transfer the DNA-containing solution to a sterile 1.5-???? mL centrifuge tube. Make sure the tubes are labeled with the appropriate TRU- Sample #, followed by the letter “H” (for hair). For example, if you are processing TRU-89, you should label the top and side of the tube “TRU-89-H”.

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26.???????? Place the tubes (now containing the extracted DNA) in your personal freezer box and place the box back in the freezer.

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Clean-up:

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1.???????????? After you are done, be sure to put your DNA extraction kit away.

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2.???????????? Then discard all debris (used tubes, gloves, used toothpicks, etc.) in the trash.

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3.???????????? If you used up any items (e.g. beaker of sterile 1.5-mL tubes, box of filter tips), ?????????? replace them from the supply shelf for the next person.

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4.???????????? Be sure to??turn off the water bath??and lights and close the door firmly behind you ?? when you leave.

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Pcr Amplification Of Genes From Plasmid Dna Arinas Protocol

Stocks needed:
1) Primers, 10 ??M each oligonucleotide, in TE buffer
2) dNTPs, 2.5 mM each (10 mM total), in MQH2O
3) template DNA, 10 ng/??L, in MQH2O
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I make my own dNPTs from individual dATP, dCTP, dTTP, and dGTP. They are also available as a PCR mix, with or without a polymerase. (We may not always have the commercial mix in Sanders lab freezer, so please check this when planning your PCR)
Thaw buffer, primers, dNTPs, and template DNA at room temperature, then keep them on ice while setting up. Polymerase should always be on ice. Add polymerase enzyme last and start the thermal cycler program immediately.
Use thin-walled 0.2 mL PCR tubes to prepare the reactions as follows:
MQH2O
24.5 ??L
10 X Vent Polymerase Buffer
5 ??L
FW primer (10 ??M)
5 ??L
REV primer (10 ??M)
5 ??L
dNTPs (2.5 mM each)
5 ??L
Template (10 ng/??L)
5 ??L
Vent Polymerase
0.5 ??L
Use the following program in a thermal cycler with a heated lid (like CSB‘s Eppendorf MasterCycler):
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1. 95 ??C 1 min
2. 94 ??C 45 sec
54 ??C 45 sec
72 ??C 1 min (adjust based on the length of fragment to be amplified, approx. 1 min per 1 kb)
Go to step 2. x 29
3. 72 ??C 2 min
4. 4 ??C hold

Purification Of Plasmid Dna Protocol

Qiagen‘s QIAprep plasmid DNA purification kits are the best way to quickly isolate high-quality, pure plasmid DNA for routine molecular biology applications. The kits use silica gel-based spin columns to bind plasmid DNA. Binding of DNA is followed by ethanol wash step and elution of DNA in small volume of Tris buffer (included in the kit) or water.

QIAprep Spin Miniprep Kit

Each spin column from this kit can bind up to 20 ??g of double stranded DNA and 85-95% of the bound DNA is typically recovered in the elution step. If you are purifying a high-copy plasmid, use not more than 5 mL of overnight cell culture per spin column. If you are purifying a low-copy plasmid, use 5-10 mL of overnight cell culture per spin column.

Bacterial cultures for isolation of plasmid DNA should always be grown from a single colony picked from a freshly streaked selective plate. A single colony should be inoculated into 1-5 mL of LB containing the appropriate antibiotic, and grown with shaking for 12-16 hours. Cultures should not be allowed to grow for longer than 16 hours because the cells will begin to lyse, resulting in decrease of plasmid yield.??

A detailed QIAprep Spin Miniprep Protocol can be found in the QIAprep Miniprep Handbook, a manual which is provided with each kit.

Please consider the following before starting the QIAprep protocol:

??? Read the Qiagen protocol, including the important notes for QIAprep procedures as instructed by QIAprep Handbook.

??? Sterile pipette tips and microcentrifuge tubes should be used.

??? If you are opening a new kit box, add the provided RNase A solution to buffer P1 and add 100% ethanol to buffer PE, as instructed by the QIAprep Handbook. If you are using an already opened kit box, the bottles should be marked indicating that these components have already been added.

??? Buffer P1 should always be kept at 4??C, so please return in to this storage temperature when done.

??? Final elution should be done in elution buffer provided in the kit (EB). However, if the plasmid DNA will be used for sequencing, DNA should be eluted in sterile water because EB interferes with the chemistry of sequencing reaction.

??? After 12-16 hours of growth, harvest cells by centrifugation. Depending on the plasmid copy number, there will be between 5 and 10 mL of cell culture. Ideally, to follow the Qiagen protocol and obtain optimal yield, the pellet should be in one tube. Culture tubes can be placed in a refrigerated benchtop swinging bucket centrifuge (Sorvall Legend) and centrifuged at 3700 x g for 15 minutes. The cells can also be transferred into 1.5 mL tubes and centrifuged at higher speeds in a microcentrifuge.

??? When the cells are harvested, continue by following the QIAprep Spin Miniprep Protocol which begins with resuspension of cell pellet in buffer P1.??

The plasmid DNA should be eluted into a sterile (autoclaved) tube. The solutions used in the kit are initially sterile of course, but one kit is usually intended for at least 50 plasmid preparations, so the solutions are not sterile once they are opened. Everyone using these kits should therefore be extremely careful not to cross contaminate solutions or introduce any cells or plasmid DNA into the buffer bottles. Pipette tips should be sterile and must be changed after each pipetting step and buffer bottles closed as soon as they are no longer needed.

Once purified, plasmid DNA should be stored at -20 ??C. A single Qiagen prep typically yields 50??L of plasmid DNA solution. The concentrations of eluted plasmid DNA can vary depending on the plasmid copy number. For high copy plasmids, the concentrations of eluted DNA are usually between 150 ng/??L and 600 ng/??L (from 5 mL cell culture). For low copy plasmids, the concentrations of eluted DNA are usually between 50 ng/????L and 150 ng/????L (from ~6 mL cell culture).

Since the volume of eluted DNA is already relatively small (~50 ????L), I usually don??et divide it into smaller aliquots before storage. The plasmid DNA is very stable and, in my experience, it is not negatively affected by repeated freezing and thawing.

The concentration of plasmid DNA can be estimated by measuring A260 and using the following conversions:

A260 of 1 = 50 ????g/ ml dsDNA A260 of 1 = 40 ????g/ ml ssDNA A260 of 1 = 30 ????g/ ml ssDNA (oligonucleotide)

Estimate DNA concentration from A260:

Concentration (????g/ml) = A260 x dilution factor x 50 ????g/ml

Remove 5 ????L from the tube containing the eluted plasmid DNA (always use a sterile pipette tips when working with DNA) and mix it with 145 ????L water for a 1:30 dilution, and then use an ultra-micro quartz cuvette. After concentration measurements I discard the contents of the cuvette. The possibility of the contamination is just too great to keep the solution used to measure.

Concentration (????g/ml) = A260 x 30 x 50 ????g/ml ???? this will give you ng/????L DNA.??

Dna Methyltransferasebased Singlemolecule Mapit Assay For Mapping Proteindna Interactions In Vi

Procedure

To map nucleosome positions using MAPit, nucleosomes are first reconstituted and probed directly or subjected to remodeling in the presence of a purified ATP-dependent chromatin remodeling complex. After terminating remodeling reactions, DMTases are added to methylate accessible DNA sites. Subsequently, samples are analyzed by bisulfite genome sequencing (BGS), which reveals methylation patterns of individually cloned molecules at single-nucleotide resolution (Clark et al., 1994; Frommer et al., 1992) (Fig. 1).

Preparation of Nucleosome Substrate

  1. Recombinant core histones (Xenopus laevis)??are expressed, purified and refolded into the octamer using standard methods (Dyer et al., 2004).??
  2. Double-stranded DNA fragments ??are prepared with one Cy5-labeled and one unlabeled primer in a preparative PCR reaction.
  3. The large-scale PCR reactions are then concentrated (we use Millipore ultracel YM-50 filters) and purified by phenol-chloroform extraction.
  4. After ethanol precipitation, the final concentration of DNA is adjusted to 1 mg/ml for reconstitutions.
  5. Mononucleosomes are reconstituted on a micro-scale using rapid salt dilution method. Typically, we titrate the DNA-to-octamer in molar ratios of 0.3:1.0, 0.5:1, 0.75:1.0 and 1.0:1.0 in a 10 ??l reaction containing 2 M NaCl and 10 ??g of octamer and use the sample with the least amount of free DNA for further analysis. Higher molar ratios can also be employed to achieve maximal reconstitution of free DNA.
  6. After incubating for 25 min at 37??C, the salt concentration is serially diluted to 1.5 M, 1 M, 0.7 M and 0.3 M by the step-wise addition of 25 mM Tris-HCl, pH 8.0 at 10 min intervals.
  7. The efficiency of each nucleosome reconstitution is verified by polyacrylamidegel electrophoresis (PAGE) at 4??C on a 4% native gel buffered with 0.2X TBE.
  8. An image of the gel is documented using an appropriate imaging apparatus.

Nucleosome Remodeling

  1. Yeast ISW2 complex (or other desired activities) is purified as previously described (Tsukiyama et al., 1999).
  2. Remodeling reactions can be performed as described in Zofall et al. (2004). A typical remodeling reaction of 75 ??l contains :??
    ??Reconstituted nucleosome ??~ 6 ??l (1-18 pmol)
    ??ISW2 binding buffer ??1X final
    ??ISW2 complex ??3-84 nM final
    ??10 mM ATP ??0.6-0.8 mM final

    Reactions are incubated at 30??C for 30 min and stopped by adding ATP-??-S (5 mM final).??

  3. Reactions(4-6 ??l) are analyzed by electrophoresis on a 5% non-denaturing PAGE buffered with 0.2X TBE at 4??C.??
  4. Document the gel using an instrument capable of fluorescent imaging .

Probing Mononucleosomes with DMTases

Cytosine-5-specific DMTases that recognize specific dinucleotide sites (e.g., M.SssI with CG specificity (Renbaum et al., 1990) or M.CviPI with GC specificity (Xu et al., 1998) are particularly useful because of their high mapping resolution. C-5 methylation (m5C) is required to provide a single-molecule view of DNA methylation via BGS.

DMTase treatments

  1. Aliquots of each terminated reaction are subjected to methylation in a 30 ??l reaction volume containing:??
    ??Remodeled chromatin in 1X ISW2 binding buffer ??25 ??l
    ??S-adenosylmethionine ??160????M??

  2. To perform DMTase titrations, serial dilutions of M.SssI DMTase (New England Biolabs) are prepared with the??M.SssI storage buffer??(manufacturer???s buffer in which the enzyme is stored) as the diluent.??
  3. Samples are pre-warmed to 30-37??C for 10 min.??
  4. An equal volume of each M.SssI dilution is added to the nucleosome mix prepared above (steps 1-2) at regular time intervals (i.e., staggered start) and incubated at 30-37??C for 5-30 min. In our titrations, a final DMTase concentration in the range of 0.01-0.64 U/??l was employed in various experiments. Dilutions of the enzyme can be avoided by adding the stock directly to the reactions but the samples must be brought up to similar volumes using the storage buffer.??
  5. Methylation is terminated by adding an equal volume of 2% (w/v) SDS (pre-heated to 70??C) and incubation at 70??C for 10 min.

Bisulfite Deamination of DNA

Several bisulfite conversion methods have been reported for processing cytosine-methylated DNA. Bisulfite treatment of DNA converts unmethylated cytosine bases to uracil while leaving 5-methylcytosine (m5C) chemically unaltered. Following PCR amplification of a region of interest, uracil arising from deamination is replaced by thymine and m5C is propagated as cytosine. The methylation status at every DMTase target site is then assessed by cloning of individual DNA molecules followed by sequencing. The following protocol was developed in our laboratory and can be used to achieve high efficiency of conversion of cytosine while minimizing DNA degradation, the two major concerns associated with other procedures. Commercially available bisulfite deamination kits such as the EZ DNA Methylation-DirectTM??Kit (Zymo Research D5020) or EpiTect?? Bisulfite Kit (Qiagen 59104) can also be used, but we have routinely achieved as good or better results with our protocol.

Kladde Lab Method

  1. Degassed water (dg.dH2O) is prepared the day before bisulfite deamination or conversion of the DNA samples is to be performed. A 125-ml bottle is filled completely (above the lip taking advantage of surface tension) with dH2O that has been boiling for at least 20 min. Then, screw the air-tight cap on tightly and cool the water overnight on the bench top. Use this dg.dH2O for preparing solutions used for bisulfite deamination or conversion in subsequent steps.??
  2. Solutions of??3 N NaOH??and??100 mM hydroquinone??are freshly made immediately before use. Sample denaturation buffer (SDB) is mixed in the ratio of:??
    ??dg.dH2O ??5.8 ??l
    ??3N NaOH ??3.0 ??l
    ??3 mg/ml glycogen?? ??0.7 ??l
    ??0.5 M EDTA, pH 8.0 ??0.5 ??l

  3. DNA (1-2 ng) ??is aliquoted and brought up to 20 ??l with dg.dH2O and 10 ??l SDB are added to denature the DNA. The samples should be incubated at room temperature while subsequent reagents are prepared.??
  4. Sodium metabisulfite solution (SMBS, ~5 g sodium metabisulfite in 7 ml of dg.dH2O, plus 1 ml 3N NaOH??and 100 ??l??100 mM hydroquinone)??are prepared, adjusting to pH 5.0 with 3 N NaOH.??Pre-warm the SMBS to 50??C.??
  5. Samples are denatured for 5 min at 98??C.??
  6. 200 ??l SMBS is then added to each sample, followed by vigorous vortexing and incubation at 50??C for 6 h in the dark. It is not necessary to overlay the samples with mineral oil.??
  7. DNA is then desalted with the EZ bisulfite DNA Clean-Up Kit (Zymo Research, cat. D5026) according to the manufacturer???s directions and eluted with 52 ??l 0.1X TE, pH 8.0, preheated to 95??C.??
  8. Desulfonation solution (DSS) is mixed in the ratio of:??
    3N NaOH ??7.0 ??l
    ??3 mg/ml glycogen?? ??1.0 ??l

  9. 8 ??l DSS is added, and then the samples are vortexed and incubated in a 37??C water bath for 15 min to desulfonate the DNA.
  10. 18 ??l 10 M NH4OAc and 200 ??l 95% ethanol are then added, followed by incubation overnight at -20??C.??
  11. Samples are centrifuged at 16,000g??for 20 min at room temperature to pellet the DNA.??
  12. Pellets are washed once with 400 ??l 70% ethanol (absolute ethanol diluted from 95% to 70% using 1X TE, pH 8.0 as diluent). Be careful as the pellets are easily dislodged from the tube.
  13. Pellets are air dried and resuspended in 20 ??l 0.1X TE, pH 8.0. The deaminated DNA can be stored indefinitely at – 20??C.

PCR Amplification of Deaminated DNA

After bisulfite treatment, DNA strands are no longer complementary to each other. Hence, deaminated templates must be amplified with strand-specific primer pairs that are designed according to the original guidelines of Frommer et al. (1992). Primer pairs for exponential amplification of the bisulfite-converted top strand are designated a1 and a2, whereas those for the bottom strand are designated b1 and b2. Potential DMTase target sites should be avoided and all other cytosines should be changed to thymine (or guanines changed to adenine) in these primers. Degenerate bases should be incorporated in primers (A and G or C and T, depending on the specific primer) in regions where DMTase sites cannot be avoided??. Also, restriction sites can be added to the 5?? ends of primers to enable subsequent directional cloning of PCR products.

  1. 2-4 ??l bisulfite-converted DNA is used as template in 50 ??l PCR reactions as follows:??
    ??Sigma JumpStart??? PCR buffer?? ??1X final
    ??MgCl2 ??2.5 mM final
    ??dNTPs ??0.2 mM final
    ??a1 (or b1) primer ??0.8 ??M final
    ??a2 (or b2) primer ??0.8 ??M final
    ??Sigma JumpStart??? Taq DNA polymerase ??1.25 U
    ??dH2O to volume?? ??

  2. The PCR amplification parameters are: 1 cycle of denaturation at 94??C for 5 min; 30 cycles of denaturation at 94??C for 30 sec, annealing of primers at 5??C below their calculated Tm??for 30 sec and then extension at 72??C for a time interval equivalent to 60 sec per each kilobase of the PCR product size; ending with 1 final extension cycle at 72??C for 5 min.??
  3. PCR products are checked for yield and homogeneity by electrophoresis of 1-5 ??l on a 1% agarose-TAE mini-gel containing 0.5 mg/ml ethidium bromide for 30 min at 100 V, and then visualized by UV transillumination. PCR products are purified with Promega Wizard?? PCR preps DNA Purification System according to manufacturer???s directions. PCR products are eluted from the minicolumn with 35 ??l sterile dH2O and can be stored indefinitely at -20??C.

Cloning and sequencing

  1. High-quality PCR products constituting single bands are cloned using standard recombinant DNA procedures.
  2. Chemically-competent??E. coli??(we use DH10B??? cells) are transformed and plated on appropriate selection plates, using blue/white screening to enrich for insert-positive clones.
  3. The efficiency of transformation with insert-containing plasmids can be verified by screening 5-10 clones by colony PCR or plasmid isolation followed by digestion with restriction enzymes .??
  4. Transformants bearing insert-positive plasmids are sequenced .??

Data analysis and interpretation

  1. Sequences obtained from individually cloned molecules are aligned and analyzed with Sequencher 4.2 or comparable alignment software.
  2. Depending on which DNA strand was sequenced in the cloned molecule, cytosines are scored as methylated if they present a C (strand with a2 and b2 polarity; cf. Figure 1 of Frommer et al., 1992 or G (strand with a1 and b1 polarity). Conversely, unmethylated cytosines are scored if they sequence as T (a2 and b2 strands) or A (a1 and b1 strands).??
  3. It is also important to determine the total percentage of C (or G), excluding cytosines present in DMTase target sites. This fraction is indicative of cytosines that failed to convert to uracil during bisulfite treatment. The recommended rate of conversion for all unmethylated cytosines should be in the range of 99.5-99.7% and sequences of molecules with lower rates of conversion (< 98%) should be discarded. Note that cytosines obtained in scored molecules that are not present in the reference sequence can arise from single-nucleotide polymorphisms in the original sample, or, alternatively, from mutations that occur during PCR amplification and subsequent cloning steps. Such cytosines should be omitted from the calculation of conversion frequency.??
  4. Lastly, only sequences that are distinct in their methylation patterns should be kept for the final analysis. This is because one cannot distinguish if non-distinct molecules are sister molecules that arise during the PCR amplification, i.e., are derived from a common ancestor molecule. The diversity of template methylation patterns can be assessed more accurately if the fragments are labeled with molecular barcodes prior to PCR amplification using specialized techniques (Laird et al., 2004; McCloskey et al., 2007).??
  5. During data analysis, it should be kept in mind that, as is the case when probing with nucleases, DMTases are also occluded from histone-associated DNA as compared to freely accessible linker DNA. Therefore, nucleosome footprints are generated when the bound histone complexes protect DNA against methylation. Spans of methylation protection of ~150 bp in size flanked by shorter methylated stretches of accessible DNA indicate individual nucleosome positions. However, nucleosomes tend to unwrap DNA transiently from the histone octamer by a rapid and spontaneous process termed site exposure, especially at the termini where the DNA enters-exits the nucleosome (Polach and Widom, 1995, 1996). Hence, a fraction of individual nucleosomes tend to display shorter or subnucleosomal size footprints with variable extents of methylation at termini that gradually decreases toward the pseudodyad centre. The observation of such subnucleosomal footprints increases in frequency with higher DMTase probe concentration. These products should not be omitted and taken into consideration to provide a detailed picture of chromatin mechanisms.

Materials & Reagents

5X ISW2 Buffer 125 mM NaOH-HEPES, pH 7.8??
85 mM NaCl??
22.5 mM MgCl2??
0.5 mg/ml bovine serum albumin (BSA)
M.SssI storage buffer 10 mM Tris HCl, pH 7.5??
0.1 mM EDTA, pH 8.0??
1 mM DTT??
200 ??g/ml BSA??
50% (v/v) glycerol
3 N NaOH ~ 0.4 g in an appropriate volume of dg.dH2O calculated using the equation: 8.333 X??_ g of NaOH = _ ml
100 mM hydroquinone ~ 0.04 g hydroquinone in an appropriate volume of dg.dH2O calculated using the equation: 90.827 X _ g of HQ = _ ml

Protocol For Genomic Dna Purification

The volumes given are for the number of cells harvested from 5??T75 flasks. We harvest by trypsinization, and wash the cells once??with PBS.
1. Resuspend the cell pellet in 9 ml of TE9 in a 50 ml conical tube. Add 1 ml of 10% SDS and invert to mix. Add 0.25 ml??proteinase K (20 mg/ml) and invert to mix.
2. Incubate at 48C overnight.
3. Add 2.5 ml saturated NaCl (6M), cap and shake vigorously for 15 seconds, then centrifuge at 2500 rpm for 30 minutes. There??will be a protein pellet at the bottom of the tube. Then, either:
4. A. Pour the supernatant into another 50 ml conical and add 2 volumes of room temperature ethanol. Invert several times to
precipitate DNA, and remove DNA strands by fishing out with a pasteur pipet OR (for those who prefer, as I do)??genomic DNA
B. Pour the supernatant into a 50 or 100 ml glass beaker, add two volumes of room temperature, and spool out the DNA onto a pasteur pipet whose end has been heat-sealed. Squeeze out the??purification??excess liquid by rolling the spooled DNA against the side of the beaker.
??5. Put the DNA strands into a 1.5 ml microfuge tube containing 1 ml of TE pH 7.5. (Volume can be increased or decreased according to DNA amount). If you have spooled the DNA, you can either??scrape it off the pipet as it rehydrates, (rolling the pipet between the fingers helps get the DNA off into the buffer). Or,??break off the pipet tip and leave it in the tube until later. Allow to stand at 37C 2 hours (at least).??
Solutions: TE9 500 mM Tris pH9.0
20 mM EDTA
10 mM NaCl
TE 7.5 10 mM Tris pH 7.5
0.2 mM EDTA

Pure Gene Kit Protocol

Isolation of Genomic DNA from Tissue Culture Cells
1. Add 2 x 10^7cells to a 15ml screw cap tube.
2. Centrifuge at 500rpm for 10 minutes in the clinical centrifuge.
3. Pour off supernatant and resuspend cells in remaining liquid by
vortexing briefly.
4. Add 3ml Cell Lysis Solution to the resuspended cells and pipet up and
down a few times. Solution should be very viscous. Incubate at 37oC to
solubilize particulate matter if necessary but this is not usually necessary.
This next step is optional (and in fact I never do it!)
5. Add 6ml RNAse A (10mg/ml) and mix the sample by inverting the tube 25
times. Incubate at 37oC for 30 minutes. This step may be optional.
6. Add 1ml Protein Precipitation Solution to the cell lysate.
7. Vortex vigorously at high speed for 30 seconds.
8. Centrifuge at 3,000rpm in the clinical centrifuge for 10 minutes to
pellet proteins.
9. Pour supernatant into a fresh 15ml screw cap tube containing 3ml
isopropanol and invert 25-50 times to precipitate genomic DNA.
10. Centrifuge at 800rpm for 5 minutes in the clinical centrifuge to
pellet the DNA. Check to make sure the DNA pelleted. Sometimes it
sticks to the side of the tube and doesn??t pellet.
11. Discard the supernatant and add 3ml 70% ethanol. Invert the tube
until the pellet is no longer stuck to the tube.
12. Centrifue at 800rpm for 5 minutes to pellet DNA. Discard
supernatant and allow to air dry 10 minutes.
13. Add 0.3ml TE and rock overnight to resuspend. If you’re in a hurry,
carefully loosen the pellet with a pipetman and incubate at 65oC for 1 hour.
Note: Store samples at 4oC. If necessary, the lysed cells can be stored
for up to 6 months at room temperature. Yield is approximately 500mg
from 4 x 107 cells.
Cell Lysis Solution (10mM Tris-HCl pH 8.0, 25mM EDTA, 0.5% SDS)
1M Tris-HCl
1ml
0.5M EDTA
5.0ml
20% SDS
2.5ml
H2O
91.5ml
Protein Precipitation Solution (5M Ammonium Acetate, NH4OAc, Mr = 77.09)
NH4OAc
19.27g
H2O
to 50ml

Protocol For Tail Saip Dna

DNA Isolation from Mouse Tail Snips
1. Add 10mm tail snip to 1.5ml microcentrifuge tube.
2. Add 0.6ml TES.
3. Use pestle to grind tissue (approximately 10 strokes). NOTE: these little blue pestles can be purchased from VWR. I can find the catalog number if you need.
4. Incubate at 65oC for 15-30 minutes.
5. Add 17ul Proteinase K solution (20mg/ml). Mix the sample by inverting the tube 25 times.
6. Incubate at 55oC for 2 hour with shaking.
7. Add 6ul RNaseA solution (10mg/ml). Invert the tube 25 times to mix the sample.
8. Incubate at 37oC for 30 minutes.
9. Add 0.2ml 5M NH4OAC and vortex vigorously 20 seconds.
requested protocol
10. Centrifuge at maximum speed in microcentrifuge for 10 minutes to pellet proteins.
for tail snip DNA 11. Pour the supernatant into a fresh 1.5ml microcentrifuge tube and add 0.6ml isopropanol.
12. Invert the tube 25 times to precipitate the DNA.
13. Centrifuge 15 seconds in the microcentrifuge at maximum speed to pellet the DNA.
14. Wash the pellet one time with 200ul 70% ethanol (rock the tube until the pellet is no longer stuck to the bottom of the tube).
15. Pellet the DNA by centrifugation for 15 seconds.
16. Discard supernatant and allow to air dry for 15 minutes.
17. Add 500ul TE and rock overnight to resuspend. NOTE: yields vary considerably but 150mg yield is average. Concentration will be ~ 0.3mg/ml. TES = 10mM Tris-HCl, pH 8.0, 25mM EDTA, 0.5%
SDS. 5M NH4OAC = 19.27g in total volume of 50ml.

Lamda Dna Extraction Protocol

1) Grow an overnight liquid lysate
2) Spin down 1ml of lysate to remove bacterial debris
3) Transfer 0.8ml of lysate to a new eppendorf
4) Add DNAse to 1ug per ml and incubate at RT for 15 min
Lambda DNA
5) Add 0.2ml of Tris (50mM pH 8.0) EDTA (10mM) SDS (5%) shake gently and
extraction protocol incubate 70 deg for 15 min
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6) Cool on ice and add 0.15ml 8M potassium acetate
7) Spin down the protein precipitate
8) Phenol-chloroform extract the supernatent
9) Add 2.5 vol of ethanol or 1 vol of isopropanol to precipitate the DNA

Genomic Dna From Marine Invertibrates

1) wash with 70% ethanol to remove salts – I would disrupt the
exoskeletons of the crustaceans at this stage.
2) wash with 95% ethanol to ppt nucleic acids, proteins and extract
nonpolar molecules such as pigments.
3) wash with TBS with 1mM EDTA to rehydrate and remove any small water
soluble molecules. Inclusion of RNAse A in the washes can be used to
genomic DNA from liberate RNA from the tissue (sample volume determines duration volume
and number of washes).
marine
4) Liberate DNA by digestion with Proteinase K (as per Maniatis –
duration again depends on sample volume) digest until tissue dissolves.
5) Phenol CCL3 extract, CCL3 extract and ethanol ppt the DNA.
Alternatively, you can feed the digested samples into any of the genomic
DNA isolation kits/protocols.

Preparing Yac Yeast Dna By Cscl Purification

1. Grow 3ml culture of yeast strain carrying your YAC of interest in selective media (e.g. CM URA- TRP-) @ 30 C for 1-2 overnights.

2. Add culture to 50ml YPD media in flask, shake O/N 30 C.

3. Spin down yeast in 50 ml tube in benchtop centifuge (setting #5), 2 min, wash with 3.5ml 1M sorbitol, spin down again.

4. Resuspend in 3.5ml of solution containing:- 1M sorbitol, 0.1M EDTA (pH8.0), 0.1% B-Mercaptoethanol.

5. Add 0.3ml 2.5mg/ml zymolase or 0.3ml 10mg/ml yeast lytic enzyme (cheaper but not as effective), incubate at 37 C for at least 1 hour. [It is important to check cells before continuing. This treatment should generate spheroplasts which will burst in dH2O (check under microscope). If you have a high proportion of spheroplasts continue, if not wait longer or even add more enzyme.]

6. Centrifuge in benchtop centrifuge, setting#6, 3 min. Resuspend in 3.2ml TE (10mM Tris.Cl pH8.0, 1mM EDTA).

Add:- 0.32ml of 0.5M EDTA (pH8.0), 0.16ml of 2M Tris Base (pH not adjusted), 0.16ml 10% SDS, 10ul of DEPC (diethyl pyrocarbonate). Mix by inversion, incubate at 65 C for 30 min.

7. Add 0.8ml 5M potassium acetate, chill on ice for about 1hr.

8. Centrifuge 15,000g, 4 C, 10 min.

9. Transfer supernatant to 30ml tube, add 12 ml of room temperature ethanol.

10. Pellet DNA by centrifugation at 15,000g, 15 min.

11. Resuspend in 3.0ml TE, add 3.87g CsCl, make up to 4.9ml.

12. After CsCl is dissolved (by swirling), add 0.1ml ethidium bromide (10mg/ml).

[It may be useful at this point to spin down any precipitate that has formed. This can interfere with the banding process later.]

13. Transfer to ultracentrifuge tube (1/2 X 2 inches (13 X 51 mm) quick seal type tubes is what I have been using), balance tubes and heat seal.

14. Spin in vTi65 rotor at 50,000rpm (or equivalent) at 22 C for 18hr.

15. Using UV illumination recover DNA band using 1ml syringe and 20-21 guage needle. [Cutting off the top of the tube or inserting a needle at the top of the tube may help prevent bubbles which can disrupt your band.]

16. Dilute sample with 1.5 volumes of dH2O and extract ethidium bromide by adding equal volumes of isoamyl alcohol, mixing gently and removing alcohol until colour is gone.

17. Precipitate DNA with 2.5 volumes of ethanol at room temperature.

18. Centrifuge and resuspend in 50-500ul TE depending on yield (if your spheroplast step went well then 300-500ul is more appropriate).

[Much of the above method, apart from the CsCl spin, is fairly standard for preparing yeast DNA. You may find a method that is easier but given that this has worked for me I’ve stuck with it. The method could probably be scaled down because you should end up with much more DNA than you need but in order to see the band in the CsCl I suspect you have to start with a minimum amount.]