Hello, I am working with worm genotyping recently and experienced some troubles which made me very frustrated >:(
I had been using a pair of primer for genotyping my crossed worms, usually I could always get a very nice band. But for unknown reasons, that primers no longer work well, it either gave me very weak bands or no bands. I am pretty sure I did not change anything. ( I used the same PCR machine, same program , same protocol for breaking the worms)
Anybody has the same problem before or can gave me some instructions?
Thanks in advance!
we use this protocol for single worm PCR: single once you make the lysis buffer
Lysis Buffer: 0.45% NP40, 50 mM KCl, 10 mM Tris pH 8.3, 2.5 mM MgCl2, 0.01% w/v gelatine. Start with the NP40 ampole to define final volume, dissolve gelatin in warm water and add other reagents. Filter sterilize and aliquote in 1.0 mL aliquots.
Thaw the 1.0 mL lysis buffer aliquote and add 3ul of Proteinase K (20mg/mL) to final 60ug/ml
Label strips of 200 uL tube with correspondent worm IDs. Pipete 80uL of lysis buffer + Prot K in the tube and pipete 20 uL in the lid. Pick single worm in the lid.
Spin down, incubate for 10 min in dry ice/ethanol bath.
Run Lysis program 60°C for 1 h followed by 95°C for 15 min. (Proteinase K works at 50-60°C).
this is a pretty dirty prep and it fails sometimes, so you always want to have a WT, MUt and a hetz as control. I use 2.5 ul of the lysate in my 25 uL PCR.
hope that helps,
Katlin
I had the same problem on December. My last 10 samples just didn’t run well. I tried with new primer dilutions, changing the polymerase and modifying temperatures, but wasn’t very successful. I now think is the proteinase K solution. It might be too old. So I am trying a new one.
When I had a similar problem with single worm PCR, the issue was one of the PCR reagents, so try changing those. In particular primers go off the deep end when you thaw them too much, and I think that can be the issue. Also, some primers seem to be more sensitive to this than others.
Sounds like you will probably have the problem solved by now, but good luck.
Jenny!
If you have been using the same primer tube this whole time (Freeze, thaw)
, then it is probably time to order a new one.
Additionally, if you have been Freeze-thawing your dNTPs they might be bad too.
There are ways to revitalize the primers with a short heat shock, and then a quick freeze, ,this will iron out any hairpins, or dimers that could be present.
I have had a similar situation in my lab, The primers work great for one of the research techs, but I cannot make them work. I just gave up and ordered my own primers (she used an online program to design them for her). They work fine now.