I want to clone a 2.2kb fragment in pCMV Tag 2B vector using BamHI and EcoRI. I usually do a PCR(My PCR gives 3 faint non specific bands above my desired product size). So I run a gel and then purify my band of interest. Following restriction digestion, I again elute out my product of interest and perform ligation with 1:3 and 1:5 molar vector:insert ratios. I never get any colony. (The enzymes I am using are working fine) . Any suggestion will be highly appreciated. Thank you.
Hi,
Your described workflow looks fine. Have you optimized your PCR conditions e.g. by using a temperature gradient to find the best annealing temperature of your primers? This could increase the amount of valid product to start with. Avoiding the three fade bands would allow you to use a PCR purification instead of gel extraction prior to the restriction digest. The should increase the amount of available insert.
At which temperature and how long is your ligation mix incubated? Increasing the time and decreasing the temperature to 4-16 C worked for me sometimes.
However, you cannot be sure that your restriction digest of the PCR product is complete. Have you thought about an alternative method e.g. Gibson assembly?
Good luck!
Hi Ishita,
Good suggestions from Boas, I would maybe try to sequence the PCR product if that's possible just to be sure.
I wasn't sure if you meant by not getting any colonies that you don't see any colonies at all, or never got colonies with the right ligated plasmid.
Sometimes it is very useful to check the basic things (competent cells, antibiotic...etc).
Also, you can run and gel purify your digested plasmid; maybe try dephosphrylation as well.
In case you want an alternative for your PCR insert, you could possibly try DNA fragment synthesis, they are quite cheap nowadays.
Good luck!
Hi again,
Sequencing the digested PCR product to ensure a complete digest is a great idea, but it requires a huge amount of PCR product and could be too expensive. In the end, it only reveals why the current method is not working. Changing to other cloning strategies should be more efficient.
I would definitely try Gibson assembly or InFusion cloning method - it has never failed for me even with longer and GC-rich inserts and multiple inserts. But you do need to optimise PCR conditions first and make sure you use a good proof-reading Taq (we find best results with Phusion or Herculase II)
Hello Ishita
What you can also try is after you do the transformation protocol, plate one half of the total volume (competent cells+SOC+your ligation mixture) on the same day as you transform and plate the remaining volume the next day. I've found many a positive clones doing this even though I did not get any colonies after plating on the first day! This, of course, is after assuming that all your enzymes and the competent cells are working fine.
All the best!
there is problem with restriction digestion of fragment.
Try hind3 & BAMH1 if possible
I would like you thank you all for your valuable suggestions..@Boas : I have tried all possible PCR optimizations, but the three faint bands could just not be eliminated. I have tried ligation with NEB ligase O/N at 16 degrees as well as 4-5 hrs at RT . I have also tried with NEB quick-ligase for 10' at RT.@Boas and @Julie: May be we will try Gibson assembly in future (looks promising!) @Rajaei: I did positive controls to check the basic things (competent cells, antibiotic...etc) and they worked fine.@ Indraneel : Can you please explain in details. Where do you keep the remaining volume?At which temperature?@ Manoj: I have a hind III cut site within my insert. May be for my next cloning I'll consider doing that.
Regards,
ishita
Hello! I normally keep it at the ambient lab temperature which is around 26-28 degree Celsius and just plate it the next evening!
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