I am trying to remove the GFP tag from our construct using the NEB Q5 SDM kit. My primers are designed to flank the region to be deleted and when I look at my PCR products on a gel (before the kinase ligase Dpn1 treatment) they look as I would expect, with the PCR products running slightly lower than the parental DNA. However, following KLD, transformation into XL10 gold and miniprep the products run a full 2-3Kb lower than the parental DNA (I am only expecting a 800bp difference). I have screened a lot of colonies and they are all appearing like this. Also my sequencing results show only the first 500-1000bp of my gene of interest (it’s 4kb) after which it goes straight to the region of the vector after the GFP tag. I have sent 3 different preps to sequencing and there is no consistent length of gene amplified and no obvious other complementary region to which my primers would bind. As my PCR products look fine, but miniprep products are smaller than expected I therefore concluded that for some reason rearrangement is occurring following transformation. I therefore tried to transform into Sure 2 competent cells as these are deficient in some of the pathways that cause rearrangement. However I am not getting any colonies with these cells. My construct plus vector are approx. 14Kb, which may be a little large for Sure 2. Does anyone have any suggestions of what I could try next?
Just in case anyone has come across this thread again, I just thought I would update. I found that different strains (Able K, Sure 2) did not help. In the end the solution for me was to avoid liquid cultures and incubate plates at RT. For miniprep therefore I would pick a single colonly, resuspend into 100ul sterle water or media and streak out onto a couple more plates. I would then scrape the whole plate for miniprep.
My advice if you're running into problems using mutagenesis would be to forgo that route and re-clone instead. Just PCR the gene of interest out of the vector and re-clone it. Of course, this assumes you have a suitable vector without the GFP tag available to clone into (or a restriction site you can use to digest the GFP out with prior to cloning)...
Thanks for your advice, if things don't look like they are going to work out soon I think I will try recloning. I have attempted the transformation again changing some of the conditions and now have some colonies and none on my negative control. I am now going to grow up for miniprep. For sure 2 cells would you also recommend growing at a reduced temperature?
Yeah, or even at room temperature. Don't ask me why, but in my experience the slower growth rate can prevent rearrangements. It's worth the try, right? Depending on the amount of colonies you can just try the three different options.
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