HI,
I am running through hard times getting my desired DNA fragments after gel purification. I want to knock out my GOI by homologous recombination in model system. For this, I cloned 5’UTR of GOI(1kb), selection marker ORF (409bp) and 3’UTR of GOI (1.34kb) into pUC18 vector. With all three inserts, the final size of the pUC18 is 5409bp. I will have to chop off the insert region (5’UTR-selection marker-3’UTR) from the vector and use that for transfection. My inserts are between SacI and HindIII cut sites. However, according to the plasmid map, if I digest the plasmid with only these two enzymes, that would generate two fragments, that are very close to one another, and gel purification would not be possible. So, I included a third enzyme ScaI (which has a single cut side within the vector part of the plasmid) and did a triple digestion. This would generate three bands: 2764bp, 1735bp and 910bp. From this, I will have to purify 2735bp fragment. Bottom is my reaction set up:
DNA (80ug): 175ul
10X CutSmart buffer: 40ul
SacI-HF (20000U/ml): 10ul
HindIII-HF (20000U/ml): 10ul
ScaI-HF (20000U/ml): 10ul
dH2O: 155ul
total: 400ul
I digested the plasmid ON @ 37C.
I ran a fraction of digested DNA in 1% gel to confirm complete digestion. Bottom is the picture of the gel (slide 1 ):
With the rest of the sample, I carried out gel purification. I ran the gel at a low voltage (50V) for almost 4hrs. to make sure that I get good separation between my band of interest (2.7kb) and 1.7kb band. Unfortunately, while I ran another gel with the eluted product of gel purification, I found weird bands. The 2.7kb band (the actual one that I was looking for) was very faint and saw additional band at 1.7kb as well.
Bottom is the picture of the gel (slide 2).
I have no idea what is going wrong. If every thing worked correctly, I should have seen a single band at 2.7kb region. Moreover, I repeated this twice and every time found the same thing. The other gel actually had more intense bands and I could see that the bottom band (1.7kb) is actually a doublet.
I would really appreciate any comment on how to troubleshoot this issue.
Thanks in advance!
Make fresh elution buffer. Probably when you elute the DNA from the column, your elution buffer is contaminated with enzyme. Your gel-purified 2.7 kb fragment band should at least be enriched compared to the 1.7 kb fragment, but you see an enrichment of the 1.7 kb fragment, that cannot be explained unless you have contaminated solutions. It happens in all labs, so it is customary to change basic solutions like TE buffer or elution buffer regularly (and also clean and calibrate the pipettes regularly).
On a different note, 2.7 and 1.7 are not very far from each other and when you overload a preparative gel, you can get cross contamination. I would have used a 0.8% gel, and perhaps load less DNA to prevent smiling of the bands. But as I said before, you should at least have enriched the 2.7 fragment even if you have some 1.7 fragment left, so I cannot think of another explanation as contaminated solutions (all it takes is for some student in the lab to forget to change the yellow tip once....)
I agree with Jürgen Denecke. I recommend to repeat the procedure with 1 µg of DNA. If the gel elution works as expected, scale up carefully. My guess is that one of your problems is overloading of the gel and of the column.
Hello,
If it's not contamination, I would try and heat-denature the digestion at 70 degrees before loading the gel, just in case you have some secondary structures that bind the two bands.
From your description I understand you want a linearised DNA fragment for transfection. I wonder if it would be easier to use the plasmid as template in a PCR reaction and extract the construct with flanking primers? If the template poses a downstream problem you can digest it with DpnI (it cuts only methylated DNA, so it would not affect your PCR product) and then column-purify the PCR.
Cheers,
Ruben
hi, did you ever found out what it was? I have the same with a gelpurified PCR fragment. After digestion of the gel purified fragment and the weird lower band (which was indeed also 2 bands), I just get the bands I am looking for. I was thinking it might be a secondary structure of my gene.
Hi Wendy,
In my case, the problem was the kit. Probably I had some sort of contamination into the gel extraction kit that I had earlier. Changing the kit resolved the problem.
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