Hi everyone,
I have done hundreds of transformations and I am comfortable doing it. I have never had problems with it. It is normally a 2-3 days job. Quick and easy.I am doing a project of enzyme activity. I did not have a problem of running protein gels, or test the enzyme activity using the positive control, etc. However a recent couple of days I am stuck at the step of dh5 alpha cell transformation. We use NEB® 5-alpha Competent E. coli (Subcloning Efficiency) C2988.
We ordered a construct of our interested gene in a pQE60 vector. The only thing I need to do is to transform the plasmid into our competent cells and amplify more plasmids. So I followed the protocol, thawed the cells, added 5ul of DNA, sit for 30 mins, heat shock in 42 celsius for 30 secs, sit on ice for 5 mins. Shake with S.O.C for one hour, then put 50ul on one warm amp plate, the rest the other.
The plates did not have a lot of colonies grow on them, but they had some colonies. So I picked the colonies, inoculate into Amp LB, shake 37 degrees overnight. Then did mini prep.However, when I digested the DNA from mini prep to confirm, the gel did not come out as I expected. It is larger. I was expecting 3977, the band looks around 4500. The double digest I was expecting was two bands, the result is three bands (not the undigested band though).
I thought it is probably some contaminations, so I did the transformation again. I washed the pipette with ethanol, changed tips, changed S.O.C. The result is still the same. Then I started to think probably they have sent us the wrong plasmid. So I digested the DNA the sent to us. The DNA band seems like it is the correct thing. Something weird must have happened during transformation.
Then I think even with a little contamination, the colonies of the correct DNA should still be the majority. So I did transformation again and picked 8 colonies. They are still the same weird DNA that I don't know where it came from.Now I am wondering if I have made the plate wrong? I made it with160ug/ml Amp, the recommended is 100ug. I am planning on remaking plates. I am also wondering if recombination has happened in the cells? Some bacteria genome DNA probably got in? It is rare because the competent cells are a "Reduced recombination of cloned DNA (recA1)". A professor from another lab kindly offered me another kind of competent cells. I want to do the transformation again using other cells. I wonder if you have any suggestions? Transformation should not be hard. I don't know why this is happening.
Thank you!
I will also suggest you select few colonies and do sequencing , if your have the correct insert and no mutation occurs, then you can proceed to troubleshoot or optimize using your RE. In addition I heard you said ''you ordered a construct of your interested gene in a pQE60 vector'' Do you have the complete sequence of the vector?, its important to note all the sites your RE will cut on the vector with your insert.
Best Wishes
Hi Kristin,
Thank you! Yes, we double-checked, the vector has an Amp. We have the sequence of the entire construct, I made the map on APE and designed which enzymes to use. I did uncut, one cut with BamHI, double digestion with BamHI and BgI1, double digestion with BamHI and XhoI, one cut with XhoI. None of them has the expected result. However, when I digest the DNA from the original tube they send me, the bands are as expected. I added the three bands BamHI and BgI1 cut, the total is around 4500, bigger than I would expect. I mean, when I am doing one cut, the miniprep I made looks bigger than the one cut the original tube is. I will look into colony sequencing. I would love to know what is happening here. My PI said Dicty DNAs have high TA content, it is easy for recombination to happen. Will the colony sequencing also tell us if the insert is in the right vector, too? Or it will only sequence the insert? I am done keep doing transformation now. I emailed the company and they can mail us some bacteria. I am just thinking probably I should wait until the bacteria arrives, streak it out, and see if I can get the right plasmid out of that. If not I don't know what to do. I am going to make some new plates now just in case it is because of the amp plates.
Hi Nasiru,
Thank you! Sequencing is a good option. Yes, we have the complete sequence of this construct. I have never used colony sequencing before. Sounds very convenient. I am waiting for the company to send me the bacteria they transformed. Hopefully, I could streak out the right thing. I am very alert right now since if even the transformation could go wrong, streaked out colonies could go wrong too. :)
I ended up using another competent cell XL-blue instead of DH5a and got the plasmid I wanted. I suspect homologous recombination happened between my plasmid and the genome DNA in DH5a.
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