As these are widely used primer sets a likely reason for pcr not working is either the quality or quantity of the dna template although it is possible that your thermal cycler is inaccurate. What is the amount in ng and OD260/280 of your dna and how many cycles are you running. You could try a lower annealing temperature just to get some amplification but too much dna or dirty dna can bind Mg and inactivate the enzyme so the quality of the dna is worth checking

Hi Paul,

These are my readings for my tails nips DNA (Blue Ink), and I took another reading of my PCR tubes after the gel didn't work (Red Ink).

I don't know how to interpret these data correctly, and there's some amplification of DNA, I don't know if this sufficient, and if the gene of interest was amplified or not.

Hi Ahmad,

just to check I am reading the results correctly your A260/230 values are in blue and are around 0.3-0.4. If this is the case then something is absorbing at 230 . If you are using standard dna prep kits they use a lot of thiocyanate to act as a denaturant. If you use too much crude lysate or not enough wash solution this thiocyanate can wash off with the dna and if the 260/230 is too low ( more than 2.0 is good) then this may denature the taq enzyme in the pcr leading to zero /poor amplification. If you are using a commercial kit try loading less tail tissue or doing an extra wash step before eluting your dna

I understand your point, but in my lab we are using a protocol to extract DNA from tail samples, we don't use commercial kits. We do it using these steps:

1- 200 ul NaOH on tail snips, put at 95 C for 20 minutes

2- Let it cool

3- Add 20 ul of 1M Tris Buffer pH = 8 to stop DNA extraction

4- Centrifuge at 4 C for 15 minutes at 15000 RPM

Usually this yields enough DNA for PCR Genotyping. We use for all our mice and it works fine, I don't think it's the problem. I don't have much experience with DNA other than this unfortunately.

You are right chaotrophic salts are not used in this method, High inorganic salt concentrations will also cause a low od260/230 and there is plenty of salt in this method. This will also make nanodrop measurement difficult as nano relies strongly on the blank solution being exactly the same as the dna solvent so blanking /zeroing will probably be inaccurate. Is the NaOH fresh or stored. NaOH will absorb CO2 from the air and form carbonate quite quickly unless prepared fresh.Is it possible to borrow NaOH from a colleague who is doing this currently and successfully or prepare fresh solution or as the od260/280 looks good just assay with 25-50ng dna as 2ul of your dna is approx 250ng which may have pcr inhibitors in which case less dna will give more amplification

Paul Rutland

Hi Paul, just wanted to say that it finally worked! I skipped the JAX recommendations, and used cycling settings that one of my colleagues uses on her another flox strain of mice, and it worked as expected.

Turns out it was the problem, I didn't change anything else and it worked fine, finally. Maybe JAX uses different machines that is different from our lab and doesn't work with the same settings.

Thanks a lot for your help, I learned a lot along the way and you introduced me to some new concepts.