Who can explain this? Weird cloning problem....
Posted 08 May 2009 - 07:54 AM
PCR gives the correct sized product, which I then gel purify, digest with EcoR1, and gel purify again. Ligation into the target vector (gel purified after digestion with EcoR1 and treatment with antarctic phosphatase) also seems to work fine -- seven of twelve minipreps had insert of the correct size after EcoR1 digestion... But all seven were in reverse orientation by sequencing -- perfect match of the correct sequence but all backward.
I have now miniprepped and sequenced a total of 14 colonies for one or the other of the genes, and ALL are backward.
I'm at a loss to explain this. Even if one of the cut sites is messed up on the PCR products, it should still be 50/50 which way it goes into a single-enzyme digested target vector.
Could I really be this unlucky??? Probability by chance should be 1/2^14 = 1/16,384!!
Thanks for any help.
Posted 08 May 2009 - 03:58 PM
Posted 08 May 2009 - 08:58 PM
But does this come into play if it is a cloning vector? Maybe its just by chance. Don't you think its a good idea to digest these clones in opposite orientation with Eco RI, column purify, add ligase and then transform and screen again to see if you get it in the right orientation.
This usually means your insert is toxic in one of the two orientations. Is there a promoter active from one side of your cloning site?
Posted 09 May 2009 - 11:17 AM
How many colonies are you getting on your transformation plates? How long of a non-selective growth period are you using after transformation but before plating?
Posted 09 May 2009 - 01:14 PM
The vector is MSCV-IRES-GFP and so does contain a mammalian promoter at the 5' end. Can this be a factor in creating expression/toxicity in bacterial cells? Also, both genes are human genes with no bacterial homologs. They're closely related kinases though, and so I suppose could be phosphorylating bacteria targets.
I'm using a 50-60 minute non-selective incubation before plating on LB/Amp plates, though I should say the colonies I've prepped have come from multiple plates, all representing separate ligation reactions.
Each plate comes from transforming an entire 10 uL ligation reaction (benchtop x 1hr) into 200 uL of D5Ha competent cells. I'm getting a lot of colonies (including on the vector only plate). Overall, about 1/3 of colonies I prep look correct on the gel (right size vector and insert after Eco digestion) with most of the rest being vector only (plus a few random weirdness that you always seem to get at a low frequency). One thing I've noticed, in case it matters is there seems to be small colonies and larger ones, and the larger colonies seem to be more likely to contain vector + insert.
Posted 10 May 2009 - 08:33 AM
Posted 20 May 2009 - 08:12 AM
Inserts can contain intentional or cryptic promoters that create mRNA from vector regions. For some vectors, these mRNA fragments are toxic. Lucigen (and others) place transcriptional terminators surrounding the cloning site to avoid this problem. It is unlikely that a mammalian promoter will be active in a prokaryote.
Agree! I am for sure NOT that lucky when I pick my 20 colonies and get all with the correct orientation. Your insertion provides some kind of selection. Maybe you can add a transcriptional terminator by yourself with PCR. Good Luck!