Protocol For Yale Rna Prep

Protocol for Yale RNA prep

Protocol For Single Worm Pcr

http://thalamus.wustl.edu/nonetlab/ResourcesF/WormPCR.htm

Protocol To Extract Dna From Paraffin Blocks

Credit: http://bts.ucsf.edu/chen/Protocol/Protocol%20to%20Extract%20DNA%20From%20Paraffin%20Blocks.pdf

Day 1
1. Cut 10-20X (30um) sections of formalin fixed paraffin samples into eppendorf tubes.
2. Add 1ml Xylene and incubate at RT for 15min.
3. Spin down for 5 min at 13000 rpm and discard supernatant.
4. Add 1ml of 100% ethanol and incubate at RT for 15 min.
5. Spin down for 5 min at 13000 rpm and discard supernatant.
6. Add 500ul of proteinase K buffer (50mM Tris pH 8, 1mM EDTA, 0.5% Tween 20).
7. Incubate overnight at 55°C shaker.
Day 2-5
1. Add 20ul proteinase K (stock solution 20mg/ml in water, store at -20°C) Final Conc.=0.4mg/ml
2. Incubate overnight at 55°C shaker.
(Solution will become clear. You may increase proteinase K conc. up to 1 mg/ml)
Day 6
1. Add 500ul Phenol Chloroform into tube and wait for 5 min at RT.
2. Spin down at 5 min with 13000 rpm.
3. Get off the upper layer. (we need the upper layer)
4. Mix it with 500ul of PCI in eppendorf tubes.
(Phenol-chloroform-isoamylalcohol extraction 1:1 v/v)
5. Shake gently and incubate for 10 min at RT.
6. Spin down at 5 min with 13000 rpm.
7. Collect the supernatant into new eppendorf tubes.
8. Add 300ul of 7.5M ammonium acetate, 1ml cold 100% ethanol and 5ul glycogen(stock (stock = 20ug/ml)
9. Shake gently and incubate at -20°C for 2 hours or overnight.
10. Spin down for 30min at 13000 rpm and discard the supernatant.
11. Air dry pellet and dissolve in 25ul of water or TE buffer.
Proteinase K buffer
Tris pH=8
0.5ml
0.5M EDTA
40ul
10% Tween
1ml
dd H2O
28.5ml
Total
20ml
Proteinase K dissolve solution
Tris HCL pH=7.5
0.1ml
CaCl
0.2ml
Glycerol
10ml
Water
10ml

Protocol For Simplified Dna Extraction From Cell Or Tissue

Protocol for simplified DNA Extraction from Cell or TissueCredit: http://www.protocol-online.org/prot/Protocols/Simplified-DNA-Extraction-from-Cell-or-Tissue-1157.html
Purpose
DNA extraction without phenol extraction and centrifugation.
Procedure
1. Lysis: the lysis buffer (usually 0.5ml) is added to the tissue or cell ( for a 75cm2 flask, add 5ml directly to the cell). Digestion is complete within several hours at 37C (cell, 2-3h) or 55C (tissue) with agitation.
Lysis buffer
100mM Tris Hcl pH 8.5 ……….. 5ml
0.5M EDTA …………………. 0.5ml
10% SDS …………………… 1ml
5M NaCl……………….. 2ml
20mg/ml Proteinase K ……………….. 0.25ml
Bring up to 50ml with dd water

2. isopropanol precipitation: one volume of isopropanol is added to the lysate and the samples are mixed or swirled until precipitation is complete ( about 10-20 min ) (viscosity completely gone).
3. Recovery of precipitate: the DNA is recovered by lifting the aggregated precipitate from the solution using a disposable yellow tip. Excess liquid is dabbed off and the DNA is dispersed in a prelabeled Eppendorf tube containing, depending on the size of the precipitate, 20 to 500ul to 10mM Tris HCl, 0.1mM EDTA, pH 7.5. complete dissolution of the DNA may requires several hours of agitation at 37C or 55C (may need overnight). It is important that the DNA is completely dissolved to ensure the reproducible removal of aliquots for analysis.
Note
Adopted from Laird PW, Nucleic Acids Research 1991,19:4293

Protocol For Rapid Isolation Of Genomic Dna From Human Oral Mucosa

Credit: http://www.protocol-online.org/prot/Protocols/Rapid-Isolation-of-Genomic-DNA-from-Human-Oral-Mucosa-3450.html

Procedure
1.Take an oral smear
2.Dissolve in 50 ml H2O through vigorous shaking
3.Pellet cells 5 min at 4000 rpm
4.Re-suspended the pellet in 400 μl Lysis buffer (50 mM TRIS-Cl pH 8, 10 mM EDTA, 2% SDS)
5.Incubate for 5 min at 65¡ãC
6.Add 250 μl 4.5 M NaCl and mix
7.Pellet cellular debris (13000 rpm for 4 min). Genomic DNA stays in the supernatant
8.Precipitate DNA by adding 650 μl of isopropanol to the supernatant and centrifuge 5 min at 13000 rpm
9.Re-suspend the DNA in an appropriate buffer (e.g. TrisCl, pH=8.0)

Protocol For Mouse And Human Genomic Dna Extraction

Credit: http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1169

Treat human blood and supernatants as potentially infectious. Discard supernatants by an appropriate method, eg into bromochlor solution.

DNA extraction from frozen blood:

1. Collect 10ml blood into a heparin or EDTA tube.
2.Transfer to a 50ml falcon tube and freeze overnight at -20°C.
3. Allow to thaw in iced water for several hours.
4. Add 40ml of triton/sucrose lysis buffer to blood and invert to mix. Spin samples at 3000rpm for 15 minutes. Discard supernatant.
5. Add 20ml of triton/sucrose lysis buffer and resuspend white cell pellet with a blue tip. Spin samples at 3000rpm for 10 minutes. Discard supernatant.
6. Add 3ml salting out/lysis buffer to the white cell pellet. Resuspend using a blue tip. Add 200µl 20% SDS and 300µl 5mg/ml proteinase K. Incubate samples overnight at 50°C in a gently shaking incubator. If sample has not been completely digested overnight, add 50µl 5mg/ml proteinase K and continue incubation for several more hours.
7. Add 1.5ml 6M NaCl and shake vigorously for 15 seconds. Spin sample at 3000rpm for 15 minutes.
8. Transfer supernatant to a new tube, and add 10ml absolute ethanol. Allow the DNA to come out of solution. Hook out DNA and transfer to an ependorf tube. Spin briefly and remove ethanol. Add 400µl TE and allow DNA to resuspend overnight at 4°C.
9. Extract once with phenol/chloroform and once with chloroform. Ethanol precipitate and rinse pellet with 70% ethanol. Allow pellet to resuspend overnight at 4°C.

Triton/sucrose lysis buffer1 litre:

1M MgCl2 5ml
1M Tris-Cl pH 7.5 10ml
Triton X-100 10ml
sucrose 109.54g

Salting out/lysis buffer100mls:
 

1M Tris-Cl pH 7.5 1m
5M NaCl 8mls
500mM EDTA pH 8 400µl

 


Mouse:

1. Dissect one lobe of liver tissue, and freeze in liquid nitrogen.
2. Grind tissue into a fine powder under liquid nitrogen.
3. Transfer tissue to a cold 15 ml blue top tube, add 5 ml of salting out/lysis buffer (10 mM Tris-Cl pH 7.5, 400 mM NaCl, 2 mM EDTA pH 8) and resuspend tissue with a blue tip.
4.Add 200 µl 20% SDS and 300 µl 5 mg/ml proteinase K. Incubate samples for
5.several hours at 50°C, until tissue had been completely digested.


Extraction and precipitation:

1. Add 5 ml phenol equilibrated with 500 mM Tris pH 8, place on slowly rotating wheel for 15 minutes. Centrifuge 3500 rpm for 10 minutes. Remove aqueous phase to another tube.
2.Add 5 ml phenol/chloroform equilibrated with 500 mM Tris pH 8, place on slowly rotating wheel for 15 minutes. Centrifuge 3500 rpm for 10 minutes. Remove aqueous phase to another tube.
3. Add 5 ml chloroform equilibrated with TE, place on slowly rotating wheel for 15 minutes. Centrifuge 3500 rpm for 10 minutes. Remove aqueous phase to another tube.
4. Add 0.5 vol isopropanol and allow DNA to come out of solution. Gently swirl the tube for a few minutes. (**At this stage, tube can be placed at -20°C overnight.)
5. Hook out the DNA with a pasteur pipette hook, and allow excess solution to drain from the DNA by holding the blob to the side of the tube.
6. Place hook with DNA on it into a 15 ml blue top tube with 1 ml TE. Break off pasteur pipette and leave hook with DNA in tube. Place on very gently rocking platform for several hours until DNA has completely gone into solution.
7.Quantitate and resuspend DNA at a final concentration of 500 µg./ml.

 

Protocol For Isolation Of Mouse Genomic Dna

 


Credit: http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1178
Kill mouse by cervical luxation and dissect liver. Add to a 50ml tube and place on liquid nitrogen. Grind to a powder under liquid nitrogen using a mortar and pestle.
Add powder to 10 volumes of extraction buffer:

Extraction buffer:
– 10mM Tris HCl pH 8.0
– 0.1M EDTA pH 8.0
– 0.5% SDS
– 20µg/ml preboiled RNAse A

1. Mix by inversion several times and incubate at 37°C for 1hr. Note on all subsequent steps avoid shearing. Do not vortex, shake or pipette the solution through narrow gauge pipettes.
2.Add proteinase K to 100µg/ml and mix in with a glass rod. Incubate at 50°C for 3hr.
3. Extract 3 times with phenol saturated with 0.5M Tris pH 8.0, 10mM EDTA. Each extraction should be mixed for 15min at RT slowly on wheel, spun 3K in benchtop for 10min and the supernatant removed with a large bore pipette.
4. Extract twice with CHCl3 saturated with 0.5M Tris pH 8.0, 10mM EDTA.
5. Dialyse aqueous phase against three changes of 1000ml 0.2 X SSC for 24hr.
6. Precipitate DNA with 0.1 volume of 3M Na acetate, 1mM EDTA pH 7.0 and 0.54 volumes of isopropanol. Spool DNA on a glass rod and dissolve in 10-20ml 0.2X SSC overnight.
7. Read OD260-280 and set up digests of 10µg in restriction buffer with and without EcoR1. Incubate overnight at 37°C. A continuous smear, with a prominent satellite band around 1kb, with EcoR1 and a tight band at limit mobility (>23Kb) without the enzyme, should be apparent.

 

Protocol For Immunofluorescent Staining Of Adherent Differentiated Es Cell Embryoid Bodies

Credit: http://www.med.upenn.edu/
1. Differentiate ES cells as per STD protocol except after at least 4 days in suspension, transfer to cover slides coated with 0.1% gelatin.

2. After 10 days total, cells can be stained with cardiac markers: after 15-25 days cells can be stained for smooth muscle markers.

3. To stain cells, remove media from slides and wash cells 2X with PBS +/+.

4.. Fix cells with either 100% EtOH or 100% MeOH for 10 min at -200C.
-EtOH is more gentle for Abs that dont work as well for immunofluorescent staining

5. Air dry cells for 5-10 min and then rehydrate with PBS+/+ 2X at RT for 5 min.

6. Pre-block and permeablize cells with PBS + 10% goat serum (or rabbit serum if secondary Ab is from rabbit) + 0.1% NP-40, RT for 20 min.

7. Remove pre-block and add primary Ab at desired concentration ( e.g. use sm-actin at 5 ug/ml) in PBS + 10% goat (or rabbit) serum. Incubate ON at RT in a humid chamber.

8. Next morning, remove primary Ab and wash 6X 30 min each with PBS +/+.

9. Add secondary Ab at desired concentration in PBS + serum and incubate at RT for 2hrs. with gentle mixing.

10. After 2 hrs., wash 4X 30 min each with PBS.

11. OPTIONAL-counter stain with DAPI for 1min in PBS and wash 2X 15min each with PBS.

12. Mount with Vectaseal and a coverslip–can post-fix with 3.7% formaldehyde for 15 min at RT and wash an additional 2X 10 min. with PBS——-then view under fluorescent microscope!!!

Assay For Killing Of Crypt Cells

1.5-6cm part of intestine cut in small pieces and incubated with EDTA 15mM-DTT1mM for 5min while vortexing at high speed (5 times). 2. After each vortex, discard supernatant and put fresh solution. 3. Recover the last wash, spin it and suspend it in PIPES buffer. 4.Dilute this crypt solution to get ~2000crypt/40µL and stimulate them with 10µL of LPS (1µg/mL) or PMA (100µM) for 30min at 37°C. 5. Harvest 10µL of the supernatant and incubate it with 40µL of E.Coli preparation (1000cfu) for 1h, 37°C. 6. Plate this preparation to evaluate the killing ratio between stimulated and unstimulated conditions.

Protocol For Mouse Splenocyte Preparation

1/ Cut mouse spleen into several small pices. Then grine spleen pices in metal mesh cup (if you done have one, you can grine spleen bettwen 2 microscope glass slides).

2/Remove all big connective tissue and debris.

3/Collect cell suspension in 15ml facoll tube anf leave the tube stand up about 3-5 min. The remaining debris will come down to the bottom of the tube.

4/ Collect cell suspension to other tube, erythocyte hemolysis, and use for cell proliferation test.