I am trying to purify DNA from a gel but yields are seemingly too low. I have loaded ~500ng of DNA onto my gel, run it, and cut it out and purified it using a Quigen Gel Extraction kit. I am only getting back ~5ng/ul. Should I up the amount of DNA I am loading on my gel initially and if so, how much should I load?
Hi Charles, you should be able to recover about 90% of your DNA. If you still have DNA, I would say that load 2 fold of your target concentration after purification. Therefore, you can have enough purified DNA if your kit yield is about 50% ( worst scenario). Make sure you follow all the kit steps and prepare all the kit reagents like adding ethanol before starting your experiments. Good luck
Hello Charles,
500 ng is not that much when you want to purify the DNA with gel extraction. I would recommend to use up to 1 µg, however, this also depends on the fragment size you want to extract. Smearing of DNA can be prevented or at least reduced by lowering the voltage from 80V (TAE gel) to 60V.
Your yield is extremely low indeed. This problem can be related to poor elution efficiency. You can use pre-warmed elution buffer (approx. 50 degrees C). Pipet the warm elution buffer onto the column and let it stand for two minutes. Then centrifuge and load the supernatant again onto the column. Wait again, then centrifuge. In some cases this can lead to an enrichment of eluted DNA.
Regards, Christian
how many ul of eluate are you getting?
5ng/ul could be ok if you have a large elution volume. Are you precipitating the purified dna if so a co precipitant
like glycogen or linear acrylamide will increase the yield
is this pcr product or genomic dna or plasmid/insert and if it is genomic or plasmid are you sure there is no rna present in the original sample making it look more concentrated,
Does the band on the gel look like 500ng pre purification or could the problem be further back in the measurement of the amount/purity of the dna?
Hi, 5 ng amount is enough for ligation (I assumed it was for Ligation.). I usually get 12-20 ng/ul from 50 ul gel well volume. During digestion, I would say start with 2 micro gm of DNA
It depends on how much will you need for further downstream procedures.
Exactly, it depends what you need. The more you load for purification, the more you can get from it.
For example, from single bands derived by restriction digestion to be used for cloning (by ligation) I felt weel confident by using QIAquick Gel extraction kit (Qiagen). I quantified using a Nanodrop 3300 Fluorospectrometer (Thermo Scientific) with the PicoGreenVR dsDNA reagent kit (Molecular Probes, Life Technologies).
I hope it may help.
AMC
Hi Charles,
I usually recommend 1µg of DNA for gel extraction. If this DNA concentration is diluted in too large volume to be loaded on the gel, you can bring it down to as low as 5 µl by vacuum centrifuge.
Good luck.
Firas.
I think 1 µg DNA have to be loaded in gel for better yield
If your elution volume is 100 ul (which is usual), then 5 ng/ul is 100% recovery. Congratulations!
Hi Charles, you can increase the DNA loading your gel to get more yield.
if your DNA is diluted use >3 lanes using tape in your gel loading comb. or if its from plasmid increase the concentration in yr initial experiment by lowering the elution buffer accordingly.
i would say 500 ng is too low to start, unless u have precious DNA in low volume.
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