Method I use:-
a. ~1cm tail tip is cut from the mice
b. O/N digestion of tail tissue by Tail Lysis Buffer containing EDTA, Tris, SDS, NaCl and Proteinase K.
c. Treatment with 7.5M NH4OAc
d. DNA precipitation by 100% Isopropanol
e. dehydration using 70% Ethanol
f. Air dry and resuspend in TE buffer
g. Heat at 55℃ for 15mins
h. Concentration measurement using Nanodrop (Usually 600-800ng/ul)
i. Run an Agarose gel Electrophoresis in 0.7% Agarose gel freshly prepared in 0.5X TAE buffer, for ~40mins at 100V
I usually use 1-2ug of DNA sample for electrophoresis. After gDNA extraction, I use it for genotyping. So, I run a PCR with the necessary primers. Normally, the PCR product should be visible for all the samples, with the variance in the size of the PCR product being the determining factor the genotype.
I am facing the following problems:-
1. When I run an Electrophoresis to check the quality of the gDNA extraction, I see smearing and "shooting star" like bands (Attached picture).
2. After running the PCR, some samples do not show any PCR product, which is weird because, all the samples should show a band (I use 200ng for PCR).
Please provide your insights to the above problems. Your comments will be highly appreciated.
Thank you.
Laurence Stuart Dawkins-Hall
I actually forgot to mention the Proteinase K in the lysis buffer. Final conc of Proteinase K in lysis buffer is 0.5ug/ul.And the final conc of NH4OAc is 2.5M. After adding this, I spin down the sample and collect the supernatant, avoiding the whitish substances between the sup and the tail tissue debris pellet. Then I proceed with the Isopropanol precipitation and subsequently to 70% Ethanol dehydration. Usually my Nanodrop reading has a 260/280 ratio ~1.9 and a 260/230 ratio ~2.2.
By what you said, looks like my gDNA might not be degraded, but might have some contamination from proteins and lipids. But if that is the case, it is still not clear why for some samples, there is no PCR product observed in the post-PCR electrophoresis. If you could comment on that, it would be of much help.
Thank you.
- Badges
- Science method