I have a 4.8 kb insert that I have been attempting to subclone into pcdna3.0 for months now. In pUC57 the insert seems to propagate just fine and maxipreps give good yields. The following day after doing ligations into pcdna3.0 the insert plate has about 2x more colonies than the vector plate. So presumably the subcloning is working. I pick all the clones on my insert plate and grow them up overnight in LB+Amp liquid culture. I do minipreps the next day followed by a diagnostic digest which always comes back negative. Sequencing tells the same story.
I am beginning to wonder if the insert is being lost during the overnight liquid culture. At the same time, the insert seems to do well in pUC57 under the same culture conditions so I am not sure what is going on. I am using DH5alpha.
I just need to get this insert into a mammalian expression vector for some future experiments.
Any help would be appreciated.
I feel your pain with this problem, I had similar troubles for a long time - and sadly the only way I resolved them is by trying over and over. However, I will try to give some advice that might help one way or another - even if none of this is a good explanation of exactly what you see.
You write that you pick all colonies from your ligation plate, leading me to think you don't have that many colonies per plate? I would try first of all to increase/improve the ratio so you have more clones to screen (Ideally ~100 with only 10 or so on the background plate) and higher chances to catch a positive one despite all odds.
1. DH5a should already be a good choice if you fear that the insert is lost during culturing, leading me to think that it is somewhat unlikely that the insert is really lost despite the use of this strain. If available to your lab you could try another strain of E.coli that is less "sickly". That way you might simply get more colonies on your ligation plates to to screen for.
2. Is there something else in the plasmid except the 4.8kb insert you subcloned? If the plasmid is very big, try to vary the transformation protocol to allow more time for plasmid uptake. Though if there's only 4.8kb in pcDNA this should not be an issue.
3. 2x ratio ligation plate to vector plate is okay, but not optimal. Make sure you properly prepare your backbone by digest and dephosphorylation to decrease the "background" of re-liagated or uncut plasmid. To check if either step is a problem, I recommend two control plates: Backbone in ligation mix without ligase, and backbone + ligase to check for uncut and religated plasmid separately.
4. If your competent cells are stored aliquoted, thaw one extra and pool them, then re-aliquot them into pre-chilled tubes and transform. If the aliquoting went wrong and your colony numbers are generally low, a 2x ratio might just be attributable to that. (although I presume you did it a couple of times already and likely would have noticed such variations...)
5. If not routinely done already, digest the template DNA in your PCR mix and generally use as little as possible for amplification. Worst case: you transform some of your bacteria with template DNA that got into the ligation mix (e.g. if the primer you used to check your ligation mix also binds in the pUC57 it may explain your positive PCR result)
6. If you finally get a really large number of clones, you could screen them by colony PCR before picking, saving time on mini-preps. Be aware though that if recombination is an issue, you may have a mixed population of positive and negative clones in a colony (which will always look positive in colony PCR), so a prep + digest before sending to sequencing is still a good idea.
7. BLAST the primers you use for insert amplification to check if they are even remotely similar to something in the E.coli genome or genome from which you amplify in the case of cDNA or gDNA. I had a major problem with a primer like this, got strange fragments and repeats into my clones (even though this does not seem to be the case here, and I suppose you use mini prep DNA from your pUC57 construct as template so this should not happen...).
I hope this is of some help one way or another, I wish you good luck!
Thank you Stephanie. This has been very helpful. I generally have about 10-20 colonies per plate. Background plate tends to be pretty clean and consistenly about half of the insert plate colony count-wise. I have tried to change the ligation conditions but this has not optimized the signal to noise. Have not tried dephosphorylating the vectors simply because the background is low but might consider it for the next run.
Competent cells are fine. I use them routinely in my other cloning experiments and get good counts (200-300 on insert plate). This leads me to believe that there is something intrinsic in the insert that might be the issue.
Hi Sushanth, I think maybe you could do the sequencing to make sure the sequence of pcDNA3.0 is right (expecially the multiple cloning sites). And you could try other Restriction Enzyme cutting site (not too close to each other). Add control during digestion (single enzyme digestion). You should do clonal PCR to get the positive clones rather than ligation mixture PCR. As far as I know, the longer the insert (more than 3 kb) is, the harder the ligation will be. Hope these suggetions could help. Thanks.
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