I performed PCR for genotyping but I could not amplify one of the sample while other samples is amplifying just fine... even I put + and - control, the + ctrl is amplifying well... what could be the reason?
It could be that the dna is not pure and contains pcr inhibitors, Measure the od 260/280 which should be close to 1.8.Low values means the dna has a lot of protein so too little dna, Use 50-100ng of dna....too much dna can stop a pcr reaction. Try amplifying less dna which may dilute any pcr inhibitors. If these tries fail then lower the annealing temperature 3c in case this samplle has a polymorphism underneath on primer stopping the primer from binding, If that fails use 2mM Mg instead of 1,5mM MG in the pcr mix which can counteract pcr inhibitors. Looking at your gel the sample is working weakly so you will only need a small change to get a good strong amplification
The issue is unique to that sample. Could be a simple as not spinning down the tubes after adding the DNA or as complicated as having inhibitors or poor yield.
I do see one band, the larger of the two sizes.
Great job including all of the controls since that helps rule out other potential issues (e.g. wrong thermocycler conditions, wrong primers, etc.)!
Madhukar Kamble
Paul Rutland Katie A S BurnetteDear all, thank you for your answer. I would like to add the following information.
1. m60 DNA is ~60 ng/microlitre which by nanodrop measurements is almost similar to all samples. 260/280 is 1.9 and 260/230 is 1.7
2. I made a master mix, so I excluded the PCR inhibitors in my reagents
3. This DNA is subjected to another PCR (GFP gene) and I got amplification for m60 just fine
4. This PCR is for OB/OB mice genotyping which WT will show single band in 191 bp and the mice containing ob (mutation) will show band in 123 bp posistion (ob/ob or ob/+). So whether they are WT or Mut I expect they at least will show one band
5. I repeated without changing anything and same result came :'\. I already mix and spin down etc...
6. Is SNP possible?
Making a mastermix will not remove pcr inhibitors. They are chemicals purified with the dna so are sample dependent. Lowering the annealing temperature is worth a try in case one primer sits over a SNP. Check the region for snps maybe on snpdb and mouse database, If these fail then design new primers 30 bases outside your current primers , amplify and sequence this sample but lowering Ta usually works but may need lowering by up to 6c if the snp is at ,or near, the 3' end of one primer
You could try just redoing the PCR for that one sample and the controls. I'd try diluting the DNA and using the "full-strength", 1:10, and 1:100 dilutions for M60. I would bet at least one will give you good data.
As an aside, based on how you described your PCR assay, all of your mice are heterozygous (show both bands) except M59, which is wild type.
Suhua Li That is is extremely unlikely scenario. There are only 3 samples of each and these are inbred lab mice.
Words of wisdom: don't make up unusual scenarios/mechanisms when the most likely answer is user error.
- Badges
- Science method