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.
Dear Suktika
- You make no mention of Proteinase K digestion either over night with SDS NaCl buffer or @ 55C for 1hour after lysis in your SDS buffer
- If that is the case and proteins remain that could explain the deposits in your wells (which is essentially a DNA/protein composite)
- Furthermore, you have extreme smearing in all of your samples: I notice you have added a high molarity solution of Ammonium acetate before precipitating with isopropanol
- If you precipitate with isopropanol you DO NOT and SHOULD not add any salt to your lysate as this will lead to high salt concentrations in your final precipitated pellet, explaining smearing and in part deposits in wells ( in conjuntion with undigested protein)
- What is more, before precipitating with isopropanol you need to remove residual protein and lipid by either commercial columns or phenol-chlorform or Trizol chloroform extraction
- Thus, the pattern you are seeing might not be degradation at all; rather DNA contaminated with very high levels of salt and protein
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.
- Your 260/280 ratio looks good as does your 260/230 ratio
- However, going back to what I said you need to column or Trizol chlorform purify your Prot K digested cell lysate as described above
- In addition, you load 1-2ug on a gel. This is way to mcuh DNA and in itself could cause smearing especially if the DNA has not been purified as suggested
- Try loading 200-500ng: That will give you stronger bands but at least might reduce some of the smearing you see but as Ive said I dont think over loading the gel is the sole problem; Its is over loading wil salt laden DNA that is probably causing your pattern
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