Genotyping PCR

Genotyping PCR

Summary

DNA is amplified by three steps: denaturation, annealing, and elongation. The wild type control (WT) is negative for both CRE and Cullin 3 CRE positive and cullin 7 produce two heterozygous bands wild type and knockout

Genotype PCR Theory

• KO= 1 band, shorter because we changed part of the DNA, travels a bit further
• WT= 1 band, longer because nothing was changed, doesn’t travel as far
• heterozygous= 2 different bands, one that matches the longer and shorter band
• CRE doesn’t exist in our bodies (Culling 3 does) we put CRE into the mice and it helps cullin 3. We are looking to see if the mouse has CRE or not (CRE pos or neg)

Refer to recipe for PCR master mix (scan this in)

Materials

• Forward and reverse primer
• PCR water
• Buffer
• MgCl2
• DNTP’s
• Taq polymerase
• Mineral oil
• PCR tubes

Steps for preparing/ running PCR samples

1. Warm up thermocycler
2. Ready the DNA samples to be loaded w/ pos and neg controls
3. Place PCR materials on ice (make sure taq is in the ice)
4. Grab PCR tubes and caps (enough for all samples) and label
5. Create master mix in a separate 1.7 ml tube
6. Load master mix into PCR tubes. In this example 18.5 ul of master mix
7. Load sample into PCR tube and flush with master mix. In this example 1.5 ul of DNA
8. ****add one drop of mineral oil per PCR tube
9. Place in thermocycler and run (about two hours)
10. Open method_ Slange Lab_ genotyping PCR_ run

Steps for running PCR gel

1. Once thermocycler is complete PCR tubes
2. Take out the ladder from the fridge (1 kb, blue-purple color)
3. Take out and cut the 2% agarose gels from the black fridge for your number of samples
4. In the PCR running station use the chamber labeled TAE (closest to computer) if solution level is low, fill the chamber with more TAE solution (by the sink)
5. Place your 2% gel into the chamber. Make sure you remove any bubbles formed in the wells
6. Load your ladder into the first well. Amount in this example is 20 ul
7. Load your samples and pos and neg controls. Take the pipette tip into the PCR tube (past the oil) and pull up the sample mix (should be a yellow color at the bottom of the tube) in this example load 20 ul of sample into the wells
8. After loading all samples and controls, connect the chamber to the power source (use the top grey one) and run for 20 minutes (before sample runs out of the gel)

[careful to not put hands into liquid as its running]