We recently got a new cell line in the laboratory but are having difficultly amplifying the resulting gDNA in any PCRs. We use the standard Bioline isolate II genomic extraction kit which we haven't had any trouble with for other cell lines. When DNA extracted from this cell line we get a workable yield (25ng/ul-180ng/ul depending on # cells used) and the 260/280 + 260/230 are always correct.
We have tried amplifying this DNA in >6 different primer sets which are all well optimised and consistently work for other cell lines yet it will not work for this. For reference we have completed this gDNA extraction with fresh cells on 5 separate occasions alongside other samples to rule out user error and all other samples have worked except for this one.
We have tried:
- Reprecipitating the DNA using a standard salt-ethanol protocol
- Using primer sets for a different gene
- Reducing DNA to 5ng, 10ng and 20ng (we use 50ng for standard 25ul reaction using ThermoFisher Phusion II enzyme) to dilute any PCR inhibitor
- Increasing DNA to 100ng, 200ng
- Reamplifying template (this gave non-specific bands)
- Washing cell pellet in PBS before extraction
- Extra washes during extraction process
It appears there is some cell-specific contaminant preventing PCR amplification.
If anyone has seen this before or knows of any troubleshooting recommendations that would be great!
Hmm, you could try mixing gDNA from this problematic line with a known good gDNA to see if there is indeed something in the sample itself that inhibits PCR in general.
Any chance this sample is genetically divergent from your other cell lines so you aren't getting amplification due to primers not being a good match?
Might be a bit odd but you could try a brief boil of the DNA and spin down to pellet protein contaminants.
Or, might be time to break out the complex gDNA prep protocol. I have a CTAB one for complex samples. It's for plant tissues but gets rid of sugars and other unwanted secondary metabolites. It takes 3 days but is worth the bother to have gDNA that works.
Have you tried primers for a really conserved gene like actin? Include a positive control.
You could try diluting out your DNA even more. Try a series of "full-strength", 1:10, 1:100, 1:1000. That ought to be enough to remove any inhibitors.
This might be a stupid question but is your new cell line from the same species?
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