Hello, and thanks for reading!!
I´m using a 50mM NaOH solution for extraction (95ºC 30´)
2uL of DNA for reaction
1,25 uL primers
1uL MgCl2
0,5 uL of DNTP´S mix
0,25uL Taq
to 23uL with sterile water
Find atached the imaging of the bands, as you can see there are three diferents pcr (with three different master mix) and the missing samples are the same in all three gels. Maybe the problem is the sample manipulation?
I Measured the concentration of DNA in the samples that don´t work (goes from 0,26 ug/uL to 0,34 ug/uL) and the ones that work starts at 0,43ug/uL is possible that it affects so much?
Does the NaOH solution have an expiry date?
Thanks
Hello Oscar,
Since you are performing colony PCR, make sure :
1) the colonies are fresh- - - - colonies with more of dead cells are very likely to contain DNA inhibitory proteins
2) use a very small portion of colony for the lysis
3) lysis time of 10 minutes is sufficient, followed by a brief centrifugation to remove the debris
4) the DNA degrading enzymes would be active at room temperature, so keeping the lysate on ice is a better choice
In your experiment, there might be an issue with the lysis. For PCR using pure isolates, concentrations of 1 ng and 10 ng are sufficient, respectively for bacteria and yeast(Refer to Molecular cloning book by Shambrook et al., for further details).
NaOH solution, kept at room temperature should not expire for a long period.
Best wishes,
Bhuwan Bhaskar
Thank you very much Bhuwan, but these are not colonies, these are mice tails.
BUT your information about NaOH and lisates will be very valuable for the next time in our lab!!
Thanks!
How many cycles of PCR are you doing? Concentration of sample makes a difference if you are not normalizing the starting quantity across all samples (e.g. performing a 100ng PCR reaction). However, your sample concentrations are fairly high, so there should be ample starting material. Some of the wells appear to have faint bands, have you tried increasing the number of cycles?
Hi Angela and thanks for your answer!
I´am doing 40 cycles, including one master mix that I usually do at 30 cycles, but due to lack of cyclers I decided tot put it together in the same cycler with the dominating protocol so some samples already had more cycles than needed.
This is a protocol I´have made hundreds of time with no problem at all.
Yesterday I tried just the non appearing samples, extracting DNA again with the rest of the tail I reserved and I made new extraction buffer... it didn´t just work again.
I think that the one who collected the samples didn´t take it on ice for a long period so DNA has degraded ... I don´t know.
WE ALREADY KNOW WHY!
They cut some of the tails after perfunding it with PFA 4%.
The NaOH extraction don´t work with fixed due to crosslinking.
NaOH does absorb CO2 from air and forms sodium carbonate over time so use fresh.
You may have pcr inhibitors so try using more Mg. Your dna concentrations seem very high 340ng/ul and 2 ul used .Check your dna concentrations. I have just read your last answer so ignore everything except the bit about CO2
Yes!! I was losing sleep for this!!
thanks for all your time and answers!
best regards!
Òscar.
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