Help -> Failed Transformations - (Jul/31/2007 )
Oh woe is me. I am struggling with my sub-cloning and time is running out. I need to move on to 2-hybrids as soon as possible, but I can't seem to get by the roadblock that is sub-cloning.
Here is problem #1:
I am trying to clone in a human CDS fragment from a CMV-SPORT6 vector, into pM and pVP vectors for the 2-hybrid analysis. Thus far, I was able to pick colonies and do mini-preps, but my results were such that I had empty vectors in all 24 samples. PI thought this was due to the pM and pVP vectors being supercoiled and the ligation didn't work.
I am re-attempting a standard ligation of 4:1 (insert:vector), overnight at 16degC. I have re-cut the vectors to ensure there is no supercoiling. The vectors are prepared Mlu1/Klenow/HindIII. The insert is prepared SgrA1/HindIII.
Any tips to ensure I get clones with the insert?
Here is problem #2:
I am trying to do a site-directed mutagenesis of a plasmid to get rid of a stop-coding amino by changing one of the nucleotides. I have complementary oligos that run 15 each way off of the nucleotide we want to change. I use Pfu Poly in the PCR rxn.
After DpnI digestion, we get three weird bands. The insert of interest (291aa/873nt) is in a pVP vector cut by NheI. The bands we get are around ~975nt, ~800nt, and ~500nt. After transforming DH5alphas I get no colonies.
What can I do to get this mutagenesis to work?
With the first ligation, Mlu1 can be tricky sometimes, You have to heat inactivate it or you would actually be carrying it on.
I am not sure how well the SgrA1 would cut. That could be the tricky one for the insert.
For problem 2,
do you have high efficient cells. Also does your vector have GC rich regions, in which case, try adding DMSO, which should help. Also you the recommended amounts of Dpn1. More can reduce transformation efficieny.
good Luck !!!
could you use a restriction enzyme other then MluI and SgrA1. I find the more exotic an enzyme the worst it cuts. How big is your insert? If changing enzyme is not an options, perhaps one might consider PCR amplifying the insert, with primers that carry restriction site more condusive to ligating into your vector.
Re-tried the whole process, from preparing the vectors to ligation to transformation to mini-preps, and again no luck. The mini-preps show empty vectors, with no insert. The control shows that the digestion of the mini-prep occurred. Back to the drawing board I suppose...
Unfortunately, the options for restriction enzymes are limited. The Mlu1/SgrA1 strategy was the best we could do, considering the pM and pVP vectors that we are trying to deposit the insert in to. The insert is around 1.5kB, so it isn't huge.
I may try the PCR amplification of the insert as a Plan B.
best of luck.
And do remember add enough skirting around the restriction site on the primer. All restriction enzymes require several base pair around the restriction site (for the enzyme to sit on) to cut said site. Most enzymes are happy with 6bp on each end of the restriction site. But some like Not and Nde require at least 8bp, any less and Not won't cut. Look up NEB technical guide on their website. Cutting close to ends.
PCR might be a good way to avoid using weird enzymes. And as perneseblue suggested, take care about how many bases you add on each end.
Good Luck !!!
Good Luck !!!
Thanks for the assistance. I have tried the addition of EcoR1 to one of my stubborn constructs, and it appears to be working. Thanks for the invaluable assistance everyone. Aside from figuring out these sorts of concepts on your own, do any of you consult a molecular lab reference text for any of these sorts of issues? If you could suggest a title or two, that would be fantastic (I'm an undergrad and I want to impress my P.I.!)
Second question...do any of you know of any good sources (forums, books, etc.) for people who are interested in pursuing graduate school? I am juggling MD vs. MD/PhD vs. PhD and I need a little more insight into the world of a PhD.