Developmental Biology

Differential Staining Of Live And Dead Embryos Using Fluorescein Diacetate And Ethidium Bromide

Materials

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Ethidium??Bromide (EtBr; Fisher)

Fluorescein??Diacetate??(FDA; Sigma)

DPBS

Acetone

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Procedure

??1)????????????Make following stock solutions:

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EtBr??(10 mg/mL??DPBS)????Store??in dark at 4?????C

FDA (5 mg/mL??acetone) Store in dark in glass container at -20?????C

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Storage life of stocks ~4 months

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??2)????????????Just before use (i.e., ~10 min) prepare the following in a 15??mL??conical tube covered with aluminum foil:

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100????l??EtBr??Stock (0.05 mg/mL)

??????3????l??FDA Stock (0.005 mg/mL)

??10??mL??DPBS or culture medium

Note:????If you want to recover embryos from glass slide, use DPBS with 0.1% BSA or serum to prevent them from sticking to slide.

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??3)????????????Place on ice until needed.

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??4)????????????To stain embryos or??oocytes??place 50????l??of dye solution on a glass slide.

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??5)????????????In the smallest volume possible transfer embryos or??oocytes??to be stained in the 50????l??and allow to sit in the dark for at least 3 min (FDA cleavage of acetate radical traps dye inside cell; 3 min=time for accumulation).

??6)????????????View embryos or??oocytes??for staining using the fluorescence microscope under UV??epiluminesence??(use UV filter).

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Live Stain=Green??????????

Dead Stain=Red/Orange

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Count??green??first before it “burns out” from illumination

Count Cell Number Of Periimplantation Embryos

Introduction??
The following is a simple procedure for determining cell number in??preimplantation??embryos. Note that DAPI has higher??photostability??than Hoechst 33342.

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Other DNA-binding dyes can also be used including??propidium??iodide and??ethidium??bromide. Staining with??propidium??iodide is a little more complicated because the dye also stains RNA and an??RNAse??step is often added.????The Molecular Probes??website has much useful information on the properties of various DNA binding dyes.

Preparation of Embryos??
1.??Remove embryos from embryo culture medium and wash 2 times in 100 ??l drop of PBS containing 1 mg/ml??polyvinylpyrrolidone??(PVP) by transferring the embryos from drop to drop.

2. (Optional) Fix embryos in 100 ??l drop of??paraformaldehyde??solution [4% (w/v) in PBS, pH 7.4] for 1 h at room temperature. Wash the embryos 3 times in 100 ??l drop PBS/ PVP by??transfering??the embryos from drop to drop.

?? Preparation of Hoechst 33342 dye??
(all solutions are made in light-proof tubes – wrapping in aluminum foil is sufficient)

Stock 1: Dilute 25 mg of Hoechst 33342 (Sigma B2261) in 2.5 ml of distilled water. The concentration of this solution is??10 mg/ml. Store at 4 C.

Stock 2: On the day of use, dilute 5 ??l of Stock 1 solution in 10 ml of PBS containing 1 mg/ml??polyvinylpyrrolidone. The concentration is??5 ??g/ml.

Working Solution: Dilute 200 ??l of the Stock 2 solution in 800 ??l of PBS-PVP for a final concentration of 1 ??g/ml.

Preparation of DAPI??
(all solutions are made in light-proof tubes – wrapping in aluminum foil is sufficient)

Stock solution: Dissolve 0.1 mg DAPI in 10 ml??deionized??water, saline, PBS or??dimethylformadide.??Store at 4 C for 6 months.????Note: It takes several hours for DAPI to dissolve into solution.

Working solution: On the day of use, add 100 ??l stock??solution??to 900 ??l PBS/PVP. Use once and discard.

Staining the Embryo??(this step should be performed with the lights dimmed)??
1. Transfer embryos to a 50 ??l??microdrop??of working solution.

2. Stain the cells for 10 minutes.

3. Wash embryos two times in 100????l??drops of PBS-PVP transferring the embryos drop to drop.

Mounting??Embryos to Slides??
1.??Prepare clean microscope slides by dipping in a??1:10??poly-l-lysine solution (Sigma P8920) for 2 minutes. Allow the slides to dry.

2. Transfer the embryos to a poly-l-lysine coated slide in a minimal volume and allow embryos to dry for 15 minutes at room temperature. Use a diamond pen to scratch a circle on the bottom of the slide around the area where the embryo is located.

3. Add a minimal volume (2-16 ??l) of??antifade??solution (ProLong??Antifade??Kit; (Molecular Probes P-7481) over the area where the embryos were fixed.

4. Put a cover slip over the slide and allow it to dry for 2 hours in a dark compartment before counting the cells under the fluorescent microscope with a UV filter. Nuclei will appear blue. Count quickly as fading can be rapid.


Embryo stained with Hoechst 33342.

What to Do When There are Too Many Cells to Count??
Sometimes, the number of cells is too great to count accurately, especially if embryos are at later stages of development.?? There are two methods one can use to reduce this problem.????The first is to place the slide with??coverslip??under a dissecting microscope and, while examining the embryo, press down on the??coverslip??with a pencil (eraser side) to spread the embryonic cells.

A more extensive dispersal can be achieved using the following procedure that was designed originally for doing chromosomal spreads (modification of a procedure of WA King,??University??of??Guelph). Some of the cells may break but one should still be able to count individual nuclei.

1.?? After collecting the embryo from the drop, transfer the embryo into a 1% (w/v) sodium citrate solution for 3 minutes.

2.?? Place the embryo onto a clean glass slide in a minimum volume.?? Let the drop all but completely dry.

3.?? From a height of about 6 inches to a foot, drop 1-2 drops of freshly prepared 1:1 acetic acid: methanol vertically onto the embryo.

4.?? Mark a circle on the back of the slide to?? indicate??the position of the embryo.

5. Let the slide dry and then stain with nuclear dye as usual.

Genomic Pcr For Alleles Of Mouse P45 Nfe2

Prepare genomic DNA from tail biopsies, and resuspend in 150 ??L sterile ddH2O.
Heat DNA for 15-30 minutes at 65??C to dissolve.
PCR Reaction: 10X Buffer 2.5 ??L
dNTP mix (5 mM each) 1.0
Taq 0.25
NC 26 1.0
NFBS07 0.5
Neo 504 0.5
template DNA 2.0
ddH2O 17.25
total volume 25.0 ??L
PCR Program: 95??C x 1 min.
94??C x 30 sec, 55??C x 1 min, 72??C x 2 min for 35 cycles
72??C x 5 min
4??C
Resolve PCR products on 1% agarose gel and visualize under UV.
Primers Neo504 and NC26 give a band for the mutant allele at 1.5 kb.
Primers NFBS07 and NC26 give a band for the wildtype allele at 530 bp.
Primers: NC26 5??? AAC TTG CCG GTA GAT GAC TTT AAT 3???
NFBS07 5??? CAC CAA ATA CTC CCA GGT GAT ATG 3???
Neo504 5??? GCC CGG TTC TTT TTG TCA AGA CCG 3???
**All primers are 50 uM**

Culture And Transfection Of Adherent Cells Fibroblastsepithelial Cells

DMEM (5 ??C/cold room)
Dulbecco’s Modidied Eagle Medium
(500mL) 11995-065 Gibco
L-glutamine (-20 ??C) L-glutamine (200mM) 25030-081 Gibco
Pen+step (-20 ??C) Penicillin-Streptomycin 15140-122 Gibco
FBS (-20 ??C) Fetal Bovine Serum (500mL) sh-30071.03 Hyclone
Cell/Tissue culture grade dish large Tissue Culture dish 353025 Falcon
Cell/Tissue culture grade dish small Nunclon TC dish 150079 Nunclon Delta
Sterile disposable pippettes 2mL serological pippette 2mL 357507 Falcon
Sterile disposable pippettes 5mL serological pippette 5mL Falcon
Sterile disposable pippettes 10mL serological pippette 10mL Falcon
Sterile disposable pippettes 25mL serological pippette 25mL Falcon
Falcon 15mL tubes 15mL Polypropylene Conical tubes 352096 Falcon
Hanks Buffer Hanks Balanced Salt Soln. 14170-112 Gibco
Trypsin Trypsin-EDTA 25300-054 Gibco
A. Thawing and initiating cell cultures
1. Prepare cell culture medium (DMEM+) (DMEM w/ 10% FBS, 50 U Pen-Strep.,
and 2 mM L-glutamine): Add 5 mL Pen-Strep stock, 5 mL L-glutamine stock, and
50 mL FBS to 450mL of DMEM.
2. Pre-warm DMEM+ in 37 deg C water bath.
3. Prepare tissue culture-grade dishes for cells by adding DMEM+, 25mL/large dish
or 10mL/small dish.(Perform under hood using sterile technique).
4. Prepare 15-mL Falcon tube for wash by adding 10 mL of pre-warmed DMEM+.
5. Obtain cells from liquid nitrogen tanks. (Location of cells described in liq. N2
binder) and thaw vials quickly in 37??C water bath until tiny piece of ice remains.
6. Transfer cells into a 15mL Falcon tube containing DMEM+ and wash by
centrifugation for 5min at 1000 rpm. Decant supernatant by vacuum aspiration
and add fresh DMEM+ to a final volume of 2mL/large dish (i.e. if you plan on
making 3 large dishes fill to a total vol. of 6mL).
7. Add 2mL of cell suspension to each dish with DMEM+. Swirl dishes slowly in a
figure-8 path for 1 min.
8. Incubate in TC incubators (37 deg C, 5% CO2).
Notes:
1. Use sterile technique: All handling in laminar flow hood with sterile disposable
pipettes.
2. In our lab???s current stocks, 1 tube of 1mL of cryopreserved cells may be cultured
in 3 large dishes or T75 flasks (2mL/dish of washed cells). The size of the dish
and the amount of cells aliquoted depends on how many cells you want and the
amount of time the cells may grow before achieving the desired density.
3. Bring all soln. At least to R.T. (if not 37 deg C) before exposing to cells.
4. Avoid cells overshooting confluence or acidifaction of medium (clor changes to
yellow).
B. Splitting Cells (Ideal when cells cover 60-70% of dish surface area, although this
varies widely by cell type.) (Cell quality is usually maintained for 10-12 splits, or
about 1.5 months in continuous culture. Beyond this interval, it???s best to thaw
fresh stocks.)
1. Aliqoute 20mL of DMEM+ into # of large dishes corresponding to how many
times you want to divide cells. (e.g., Split 1 large dish of 293 cells @ 60%
density into 5 large dishes if you wish to harvest again in ~2days).
2. Wash Cells in Hanks??? Buffered Salt Solution (1XHBSS) or 1xPBS to remove
serum, which acts as a protease inhibitor.
3. Treat w/ Trypsin, which detaches cells from the dish and from each other.
– Remove wash Buffer by vacuum aspiration.
– Add Trypsin-EDTA to cells (3mL/Large dish, 1.5mL/small dish)
– Swirl and tap dish to detach cells (usually 2-10 min at 37??C or longer at
RT). Avoid prolonged trypsin exposure, which can be severely damaging
to cells, by periodic visual monitoring.
– After cells are fully detached, add vol.of DMEM+ so the total volume is
divisible by the number of total new plates desires 2mL/plate
(ie Add 7mL DMEM to large plate w/ 3ml of Trypsin so the tot. vol. is
10ml that will be divided into 5 new large plates (2ml/plate).
4. Mix plate by swirling and distribute cells into new plates w/ DMEM (2mL/plate)
5. Label dishes with type of cell, date split, and # of times split (Passage #).
6. Resume culture in 37??C tissue-culture incubator.
C. Lipofectin Transfection (Cell density on plates should be about 60%)
1. Prepare plasmid DNA in sterile 1mL Eppendorf tubes, Vtot/tube ~600 ??L
(1 tube will be used for 1 large plate; 20ug of DNA is usually sufficient to
transfect cells in 1 large dish or T-75 flask)
DNA stocks: Ci Cf Vf (uL)
DNA eg 2??g/??L 20 ??g 10
DMEM-/- 590
2. Prepare Lipofectin stock soln. in Falcon tube
3 ??L of lipofectin is required for 1 ??g of DNA.

Purification Of Rna With Trizol Reagent

TRIzol reagent is a mixuture of phenol and guanidine isothiocyanate suitable for small-scale extraction of RNA. (TRIzol reagent is a registered trademark of Molecular Research Center, Inc.)

1. Grind 30-100 mg of frozen tissue in a prechilled microfuge tube with prechilled micropestle. Keep the tissue frozen with liquid nitrogen.

2. Add 600 ul of TRIzol reagent (Invitrogen) and grind at room temperature until no visible debris remains.

3. Store the tube on ice until all of the samples are ground.

4. Add 400 ul of TRIzol reagent to each tube. Store the samples for 5 minutes at room temperature.

5. Add 0.2 ml of chloroform per 1 ml of TRIzol reagent. Shake the tube vigorously for 15 seconds.

6. Spin for 15 minutes at 4 C. Transfer the aquaous phase to a new tube.

7. Add 250 ul of isopropanol and 250 ul of precipitation solution (0.8 M sodium citrate, 1.2 M NaCl). Mix well. Stand the tubes for 10 minutes at room temperature.

8. Spin for 10 minutes at 4 C. Discard the supernatant.

9. Rince the RNA pellet with 1 ml of 75% ethanol. Spin for 5 minutes at 4 C. Discard the supernatant.

10. Dry the pellet for 10 minutes. Dissolve the RNA in RNase-free water.

11. Store at -70 C.

Typical yield is 20-50 ug of RNA from 100 mg of??Arabidopsis??tissue. However, yield of silique RNA is very low with this procedure in our hands. For silique, follow the protocol described bellow.

Extraction of RNA from silique

1. Make 1:1 mixture of extraction buffer (1M Tris-Cl (pH 9.0), 1% SDS) and phenol-chloroform. Phenol-chloroform is made by mixing equal volumes of TE-saturated phenol and chloroform just before use.

2. Add 30-100 mg of frozen silique into 600 ul of the above mixture in microfuge tube.

3. Grind the silique with micropestle.

4. Vortex 30 seconds, and then spin at 14,000 rpm for 5 minutes at 4 degrees. Transfer the aquaous phase into new tube.

5. Extract once more with phenol-chloroform, and then extract 3 times with TE-saturated phenol.

6. Add 1/10 volume of 3M NaOAc and 3 volumes of ethanol. Spin for 10 minutes at 14,000 rpm at 4 degrees. Discard the supernatant.

7. Rinse the pellet with 1 ml of 70% ethanol. Spin for 5 minutes. Discard the supernatant.

8. Dissolve the pellet in 150 ul of RNase-free water. Add 50 ul of 8M LiCl. Mix well.

9. Store on ice overnight.

10. Spin at 14,000 rpm for 10 minutes at 4 degrees. Discard the supernatant.

11. Rinse the pellet with 200 ul of 2M LiCl. Spin for 5 min at 14,000 rpm. Discard the supernatant.

12. Rinse the pellet with 500 ul of 70% ethanol. Spin for 5 min at 14,000 rpm. Discard the supernatant.

13. Air-dry the pellet for 10 minutes. Dissolve the RNA pellet in RNase-free water.

Typical yield is 20-50 ug of RNA from 100 mg of??Arabidopsis??silique.

Gel Shift Emsa Protocol

There are multiple variations to this protocol, but we find that this one works well in all cases we tested.

Reagents:

5X EMSA Buffer:

50mM HEPES (pH 7.9)

375 mM KCl

12.5 mM MgCl2

0.5 mM EDTA

5 mM DTT

15% Ficoll

32P-labeled oligonucleotide probe

polydI/dC:??1 mg/ml in TE

BSA:??10 mg/ml in TE

5% Polyacrylamide gel (30 ml)

22 ml water

3 ml 5X TBE

5 ml 30% Acrylamide

0.210 ml 10% Ammonium persulfate

10-100 ml TEMED

5X TBE (1 L):

54 g Tris base

27.5 g boric acid

20 ml 0.5 M EDTA (pH 8.0)

Reaction:

Combine the components in the following order (in ml):

5X EMSA buffer:???????? 4

Water:???????????????????????????????????????????? ?????????????????????? to a final volume of 20 ml

PolydI/dC:?????????????????????????????? 1

BSA:???????????????????????????????????????????????????? 1

32P probe:???????????????????????????????? 1 (10K cpm)

protein:???????????????????????????????????????? 1-3 ml

let the reaction stand for 10-15 min at room temp., then load 18 ml per lane on a 5% polyacrylamide gel.?? Run at 150 V for 2h at room temperature, then dry the gel and expose 4-16 h to film at ???80 C.

Transfast Transfection Protocol

Transfection protocol for 6-well dish

  1. Seed cells at appropriate density (5×104/well for 3T3, MPAC, BHK; 5×105/well for aTC1.6, bTC3) one to two days before transfection.

  2. Combine total DNA at 1ug /well with 600ul/well serum-free medium in an eppendorf tube

  3. Add 1.5ul/well Transfast (Promega) to the DNA/medium mixture. Vortex for 10 seconds

  4. Incubate at RT for 10 to 15 min

  5. During the above incubation, aspirate medium from cells and add PBS; remove PBS just before adding DNA in step 6.

  6. Add 600ul DNA mixture from step 3 to the appropriate well.

  7. Incubate at 37??oC incubator for 1 hr.

  8. Add 4ml regular growth medium to each well??

  9. after 24 hours, change to fresh medium

  10. After 48 hrs, harvest cells for functional assay

24 H Subcloning Protocol

The following protocol is designed for subcloning inserts (I) from one vector into another vector (V).?? The inserts can be anywhere from 30 bp to 8 kb (possibly higher).

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Perform restriction digests of (I) and (V) to obtain compatible overhangs (even blunt ligations work well with this protocol).?? Generally, perform restriction digests as follows:

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DNA??????????????????????????????????????????????????????????????1-10 ul??(2 ug TOTAL)

10X Buffer??????????????????????????????????5 ul??(determine appropriate buffer from NEB catalog)

Enzyme 1??????????????????????????????????????1-2 ul??(20-40 U is sufficient)

Enzyme 2??????????????????????????????????????1-2 ul??(same as above)

100X BSA????????????????????????????????????????1 ul

Water???????????????????????????????????????????????????????? to??50 ul total volume

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Note:?? Add enzymes LAST.?? Digest at 37 C for 2-3 h (no longer!).

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1.?? OPTIONAL:?? to the (V) digest, add 1 ul of Calf Intestinal Alkaline Phosphatase (CIP)???Boehringer Mannheim, 1 U/ul.?? Incubate at ROOM TEMP for an additional 30 min.

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2.?? During the incubation, prepare a 1-2% agarose gel in 1X TAE (NOT TBE!) with wide lanes (8-10 mm wide).?? DO NOT add EtBr to the agarose gel (otherwise this will mutate your DNA samples).

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3.?? Add 10 ul of 6X agarose gel loading buffer to the digests, and load 30 ul of the digests into a lane in the agarose gel.?? Load 7 ul of 1 kb DNA ladder in a separate lane.

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4.?? Run the gel at 70 V for 30 min to 1 h.

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5.?? Stain the gel in a 2.5 ug/ml solution of EtBr for 5-10 min (higher the percentage gel, the longer the stain time).

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6.?? Run the gel again at 70 V for 10 min (this will rapidly destain the gel).

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7.?? View the gel at LOW INTENSITY on the UV gel box, and take a 2.5 sec. Exposure picture for your records.?? Minimize exposure of the gel to UV light.

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8.?? Using a CLEAN blade for each fragment, excise the desired fragments with??as little excess agarose??as possible, and place in a preweighed and labeled eppendorf tube.

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9.?? Weigh the agarose gel slice, and add 3 volumes of buffer QG (from the Qiagen Gel Extraction Kit).?? Incubate at 42 C for 10-15 min until the gel slice is completely dissolved.

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10.?? Add 1 volume of isopropanol, mix, and load on a PURPLE spin column.?? Spin at top speed for 30 sec. And discard the eluate.

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11.?? Wash the column with 500 ul of buffer QG, spin, then wash with 750 ul of PE.

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12.?? Spin a final time to remove all traces of PE.

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13.?? Elute the DNA with 50 ul of WATER.

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Proceed to LIGATION:

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LIGATION:

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14.?? Boehringer Mannheim Rapid Ligation Kit.?? Thaw DNA dilution buffer and T4 ligase buffer quickly and place on ice.

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15.?? Add 2 ul of (V) and 2 ul of (I) to 1 ul of 5X DNA dilution buffer in an eppendorf tube.

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16.?? Add 5 ul of 2X Ligase Buffer, flick to mix, and add 0.5 ul of Ligase. ??Mix and incubate at room temp for 5 min.

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17.?? Place on ice, and proceed to TRANSFORMATION.

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TRANSFORMATION:

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18.?? Quickly thaw a tube of DH5a and place on ice.?? Prechill an empty eppendorf on ice, and add 90 ul of the DH5a into this tube.

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19.?? Add 8 ul of the ligation reaction to the DH5a, and incubate on ice for 30 min.

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20.?? Heat shock at 42 C for 45 sec.

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21.?? Cold shock on ice for 2 min.

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22.?? Add 900 ul of LB and incubate at 37 C with shaking for 30 min.

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23.?? Spin tube at 5K rpm for 2 min.?? Remove all but 100 ul of the LB and resuspend pellet.

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24.?? Plate pellet on LB plate with appropriate antibiotic.

25.Incubate inverted at 37 C overnight

Nomenclature For Mud Insertions

A variety of phage Mu derivatives (Mud phages) have been used as tranposons for formation of operon and protein fusions. Recently it has been possible to substitute one sort of Mu derivative for another by recombination events internal to the Mud phage that do not alter the insertion site or the flanking sequences in the host chromosome. This has given rise to some problems of nomenclature. We propose the following conventions to solve these problems.

1. The allele number of a Mud insertion is not changed when the nuture of the inserted material is altered by recombination with other Mud phages.

For example: If his-9887::Mud1 is isolated and assigned a his allele number, it retains this number when a KanR determinant is placed in the inserted material. Now it becomes his-9897::MudK (see below for naming of MudK).

2. Since a variety of Mud phages are available and these names fit no apparent uniform system, we propose that a shorthand name be assigned to each phage and be used in naming mutations. Below is a list that includes the Mud derivatives currently in use in our lab.

Shorthand Old Name Ref. Definition
Mud1 MudI (AmpR,Lac) [3] Original operon fusion phage of Casadaban
Mud2 MudII (AmpR,Lac) [2] Protein fusion phage of Casadaban
MudA Mud1-8 [6] Hughes derivative of Mud1 with suppressable transposition defects (operon fusion)
MudB Mud2-8 [6] Hughes derivative of Mud2 with suppressable transposition defects (protein fusions)
MudC MudII4042 [5] Automatic cloning Mud with DNA replication origin (protein fusions)
MudD mini-Mud [4] Intact lac operon including I, Z, Y, and A (no fusions)
MudE mini-Mud [4] Intact lac operon including I, Z, Y, and A in orientation opposite to that in MudD (no fusions)
MudF mini-Mud A KanR derivative of MudD. Still lac+ but carries KanR and a deletion of transposition functions derived from MudK (no fusions)
MudJ MudI1734 [1] A short Mud derivative carrying KanR and a deletion of transposition functions (forms operon fusions)

3. If simple mutations are added to these phages, the genotype can be added to the insertion designation.

For example: A Tn5 insertion in AmpR is available and can be added to any of the Mud phages carrying AmpR. If his-9897::Mud1 receives this Tn5 in AmpR it would be called his-9897::Mud1(bla::Tn5).

References

[1] Beatriz AC, Olfson P and Casadaban MJ. 1984. Plasmid insertion mutagenesis and lac gene fusion with mini-mu bacteriophage transposons. J Bacteriol 158:488-495

[2] Casadaban MJ and Chou J. 1984. in vivo formation of gene fusions encoding hybrid -galactosidase proteins in one step with a transposable Mu-lac transducing phage. Proc Natl Acad Sci 81:535-539

[3] Casadaban MJ and Cohen SN. 1979. Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: In vivo probe for transcriptional control sequences. Proc Natl Acad Sci 76:4530-4533

[4] Chaconas G, deBrujn FJ, Casadaban MJ, Lupski JR, Kwoh TJ, Harshey RM, DuBow MS and Bukhari AI. 1981. In vitro and in vivo manipulations of bacteriophage Mu DNA: Cloning of Mu ends and construction of mini-Mu’s carrying selectable markers. Gene 13:37-46

[5] Groisman EG, Castilho BA, and Casadaban MJ. 1984. In vivo cloning and adjacent gene fusing with a mini-Mu-lac bacteriophage containing a plasmid replicon. Proc Natl Acad Sci 81:1480-1483

[6] Hughes KT and Roth JR. 1984. Conditionally transposition-defective derivation of Mu dI(Amp Lac). J Bacteriol 159:130-137

Transduction

1. Grow overnight culture of recipient strain.

2. Dilute donor P22 phage lysate in either T2 buffer or saline to allow MOI (Multiplicity of Infection) to be less than one except in cases to two particle transduction (tranducing marker is large). Since phage survives in T2 buffer longer than in saline, T2 buffer should be used when diluted phage is going to be kept.

3. If vitamin prototroph marker will be selected, the recipient cells ought to be washed by saline or minimal salt solution, then resuspended into minimal salt solution.

4. Take 0.1ml of the overnight culture, mix with 0.1ml of properly diluted donor P22 phage lysate, then:
a. if selective marker is Tcr, or prototrophic phenotype, cells and phages could be directly spreaded onto selective plates.
b. if selective marker is Knr, or Cmr, cells and phages could be spreaded onto NB plates and incubated at a proper temperature for overnight, then printed to selective plates. Alternatively, mixtures of cells and phage could be incubated at room temperature for one hour before being spreaded onto selective plates. Preincubation of NB plates gives rise to more transductants and more repeatable results.
c. if selective marker is Apr, mix of cells and phages could be directly spreaded onto selective plates, or preincubated in tubes for one hour, or preincubated on NB plates overnight before selecting Apr. Preincubation is recommennded.
d. if selection will be done on plates containing EGTA, cells and phages have to be mixed and preincubated at room temperature for one hour to allow infection finished.
e. If selected marker is GnR (gentamycin) use 20mg/ml gentamycin in LB (NB will not work!). Also, first spread transduction to an LB plate without drug and print to LB Gn after overnight incubation.
Incubate the plates at desired temperature until transductants grow up as colonies.

5. Nonselective marker could be screened either by directly printing to screen plates or by picking colonies to patch onto the same selective plates and then to replica print to screen plates.

6. Pick a few of each desires type of transductants from original selective plates or the master plates, streak out on green plates to grow for single colonies.

7. Pick phage-free colonies (white on green plate), streak cross over a line of P22 H5 phage lysate to test the phage sensitivity of the transductants.

8. Pick phage sensitive transductants, streak out on NB plates for single colonies.

9. Test phenotype of transductants by patching colonies onto a nonselective medium (e.g. NB plate) followed by replica-printing to a set of testing plates. All known phenotype of the strain have to be tested.

10. Take cells of a patch with correct phenotype from the master plate, streak out for single colonies on a NB plate.

11. To save the strain, see the section “Saving Strains”.