PCR genotyping

9/21/05

1. PCR can be used to genotype animals once the locus of targeting vector recombination has been unambiguously determined by southern blotting.

2. Genomic DNA is prepared from mouse tails, and resuspended in a volume of 50uL. If you question the quality of the DNA it should be analyzed by gel and spec readings compared to samples that have been successfully used as PCR templates. Store genomic DNA at 4 degrees as it may shear with freeze-thaw.

3. Primers are specific to the gene of interest and are listed below. All primers are made up at 1ug/uL and must be diluted 1uL in 10uL before use. Store primers at –20.

4. Make sure the program is set on the PCR machine. All PCR programs take the following form:

denature 95 degrees 2 minutes

denature 95 degrees 30 seconds

anneal variable 45 seconds

extend 72 degrees variable

goto step 2 29 times

hold at 4 degrees for ever

5. Assemble a master mix for you reaction on the tails. Per tail you want

2uL tail DNA

1uL forward primer (remember to dilute first!)

1uL reverse primer

0.5uL Taq polymerase (other enzymes box)

8uL ddH2O

12.5uL 2x Buffer E (other enzyme buffers box)

other than the tail DNA, multiply each number above by the number of tails you have plus 10% extra (If you have 20 tails, make the mix for 22). Add those components together and mix thoroughly. Spin down and place on ice.

6. Set up PCR tubes and label them. Write down the order of the tubes, the contents of the reaction and the details of the PCR program. Aliquot 23uL master mix per tube.

7. Add 2uL tail DNA to each tube. Make sure to do a positive and negative control for each reaction. Positive is a tail of known genotype, negative can be no DNA template to make sure the reaction components are not contaminated.

8. If no band is a meaningful outcome of the PCR reaction, to ensure that PCR did not fail in a tube, run a control reaction of different molecular weight. You simply add 1uL of the F and R primers for the second reaction and cut the water to 6uL per tube.

9. Vortex the tubes (with caps tightly sealed) and spin down. Place in PCR machine and run. Runs take about 2-3 hours.

10. Make 2% TAE/EtBr gels to analyze the results of the PCR. Make sure to run 100bp molecular weight markers to validate the size of the bands.

Specific genotyping reactions:

CaRF

1. Primers: SneoF, SloxR2

bands 800bp = wt

860bp = floxed

100bp = recombined

anneal 57 degrees

extend 1 minute

2. Primers: SneoF, m6RI

bands 400bp = wt

440bp = floxed

none = recombined

anneal 57 degrees

extend 1 minute

3. Primers: BDNFcodF, BDNFcodR

band 82bp = wt

run with PCR reaction #2