I have a problem with my gel extraction. In fact I study termites. After the isolation of DNA, I amplified, but the extraction of DNA on agarose gel gives me a very small amount. I'm using the MinElute kit. Despite all the advice followed on the website, the amount is still low. For example, I have 556ng/ul departing, but I got 6ng/ul at the end. I make electrophoresis with 50 ul. The aim is in fact to make an Illumina Sequencing. I also would like to know if making a purification of the PCR product with PEG mix you can get a good result for sequencing?
Hi
1. Try eluting the DNA in low volumes (10-20 ul) in the last step of spin column based gel extraction kit (if you are using one). This will increase the concentration but the yield may remain same.
2. Better use a PCR clean up kits for amplicon purification. PEG based method is good but it results in loss of PCR products.
3. If you are doing gene specific amplification, I suggest you to re-amplify the gel eluted PCR product and purify the same before sequencing.
In the old days - actually, not that long ago - we would just spin the DNA out of the gel. One way to do this is to mince up the band (and gel) into tiny pieces and then spin through glass wool into an empty tube. Sometimes simple is better.
Try to use a different type of agarose, low melting agarose might help.
Instead of doing size selection on a gel, you could also use Ampure beads for size selection.
maybe this link from Qiagen can help:
http://www.qiagen.com/qdm/cup/gelextraction/benchtutorial?cmpid=QVenCUP140210BenchTutorialsFDKEM&utm_content=QVenPR140210+Transfection_Int&utm_campaign=QVenPR140210+Transfection&utm_source=iPost&utm_medium=email&cmpid=QVenPR140210+Transfection_Int&imm_mid=0b71eb
- Badges
- Science method