I am trying to purified a PCR product from gel extraction (2% agarose), for the purification I used PureLink Quick gel extraction and PCR purification combo kit (Invitrogen). And for the PCR reaction I used Go Taq Green Master mix from Promega.
The purification purpose is for sequence. After the purification I made a electrophoresis. The electrophoresis conditions: 100V, 30min, 2% agarose. I used blue/orange loading dye and GelRed for staining.
As a result I got the expected bands but I also have anomalous bands in the superior part of the gel, can these be form from de loading dye? or green master mix? will this affect the results form sequencing?
I am using blue/orange dye so I can say for sure that its not from dye unless you didn't contaminated your dye with any other sample. I recommend you to load some control. load dye only, and dye with water or buffer you used for DNA elution. Then you can have some idea.
Hi Paola,
What DNA ladder are you using? If the smaller bands at the bottom are around 50-100bp, these could be primer dimers from your PCR mix (you're primer concentration is too high or cycling temps are not optimal for efficient amplification of your product). This can also be the reason why your expected product has a lower band intensity than the smaller bands.
Some column-based purification kits will allow the smaller bands to pass through the binding material when you purify - so it may not have an effect on your sequencing.
Is there a reason why you're extracting a PCR product from the gel and then running another PCR on that? By doing this, you may inadvertently introduce mutations - though the fact the polymerase you're using has proof-reading capabilities reduces the likelihood, or damage your DNA sequence from UV exposure while you're cutting your bands out.
Hope it helps
John
Hi Paola,
The upper band at the superior part of your gel might be a genomic contamination. I suggest you need to troubleshoot properly to identify the source of the contamination. You can not use that for sequencing as far as you still have those superior bands after purification.
With respect to Dr John's reply
'' If the smaller bands at the bottom are around 50-100bp''
I will like to remind him that the bands are not smaller bands rather higher bands as seen from the attached gel picture.
Best Wishes
My apologies, misinterpreted the gel. Thanks for pointing that out! Nasiru is correct in suggesting it is likely to be genomic contamination.
You can run a few controls like no template pcr and also loading control when you repeat. The band could be the result of several things? Contamination of template, PCR reagents, water, purification reagents. There is also a chance of non-specific amplification (but unlikely in your case).
regards
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