I tried to extract DNA for agarose gel using the extraction kit from Qiagen. After mixing with the result with the loading dye (6x from NEB), it doesn't want to stay inside the pocket. Anyone has similar experience?
What method did you use preparing the dna. Samples do float out of the gel well if the dna was not dried enough and there is still some ethanol left in the dna sample
Paul Rutland I used bind-wash-elute according to the protocol provided by qiagen
(https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/dna-clean-up/qiaquick-gel-extraction-kit/).
The result also shows a huge jump in absorbance at 210 nm. In the washing buffer we have to add ethanol according to the protocol. Do you know how to get rid the ethanol completely then? another round of wash?
I tried this method twice now and always failed, will try another method like freeze and squeeze based extraction.
There are 3 things that you can try first Joel Christian
1 re-purify the sample using a QIAquick-, or MinElute column, or QIAEX II resin (expensive and will lose more dna)
2 incubate the eluate at 56°C for 10 min to evaporate the ethanol (quick and easy and cheap)
3 dry down the sample in a vacuum centrifuge, and resuspend the pellet in a small volume of sterile water ( slower because dissolving the dna may take a while but like 2 there is no loss of dna
I assume that this dna is either not genomic or is very old because this method has a high size cut off at about 10kb.
You might also get better recoveries if you spun down the bound dna for longer to get rid of more of the alcohol containing buffer and then elute the dna with 2 aliquots of water ( one at a time not double the volume in 1 elution to get a better yield of dna
Ive had this happen to me quite a few times.I ended up reducing the amount of DNA I was using for loading.Can happen due to pipetting error too
Charlotte Rodricks Sure, the less the amount of DNA the less loading dye we should use I think. However the ratio is always the same. In my case no matter how much/less I put, it floats up immediately. So there must be something wrong with the mixture of DNA and the dye. Maybe ethanol contamination as Paul Rutland pointed out.
I am not sure that it is due the pipetting error either (I put in the lane 6-9). I have a decent signal on the other lanes (non-extracted DNA).
Joel Christian Ive seen it happen with my samples but I still get a light band at least.
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