I am trying to genotype mice from Jax (stock #: 021877) using the Sigma Kit (REDExtract-N-Amp™ Tissue PCR Kit) but I dont get bands.
I've tried the protocol suggested by Jax (https://www2.jax.org/protocolsdb/f?p=116:5:0::NO:5:P5_MASTER_PROTOCOL_ID,P5_JRS_CODE:24559,021877) and the one suggested by Sigma (https://www.sigmaaldrich.com/technical-documents/protocols/biology/redextract-n-amp.html). Changed the primers and template concentration already. My ladder works perfectly though. I got band once, but never more
These are my primers:
GCA TGT TTC CTG TTT CGT GA Wild type Forward
GCA TAT TTG GCT TTT ACT CTG G Common
TGG TGG CTG GAC CAA TGT Mutant Forward
Has anyone ever genotyped the same mice strain? Or someone who also had problems with the Sigma PCR kit?
Would appreciate any help!
Thank you.
Carolina Scarpellini instead of using agarose gel you can use 6% Polyacrylamide gel (vertical gel electrophoresis) using 1X TBE buffer. it will give you better resolution than Agarose gel. If your amplification is low then this might help you.
What PCR condition you are using? You can increase the cycle number to some extent to see better amplification. If the concentration of the template DNA was not enough, there is a possibility of not visualizing any band.
Carolina Scarpellini
I have not any experiences with your work but your gel shows you have very weak amplification so I suggest you first of all use gradient PCR and if you have not reached use DMSO.
I suggest you use traditional tail cut DNA extraction, the DNA concentration will be higher than the kit. In our lab, we use this method to verify the genome type of our trans-gene mice, it works really well. If you want the protocol I can provide.
Hi guys, thank you for all the suggestions. I think my issue should be too much template and too little primer. Would this possibility make sense given the pic of the gel I provided? My template right now is about 300ng/uL. That is supposed to be enough, no?
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