Good day fellow researchers,
I have recently designed primers with restriction sites which I am going to use to amplify my insert and clone into a reporter vector. After reading up on TA cloning I was thinking of doing TA cloning first into pGEM T Easy and then subclone into the reporter vector. Would it be okay to still use the primers I have designed with the restriction sites to clone into pGEM T Easy and then subclone into the reporter vector? I know that I would need to use a taq polymerase (non-proofreading) for the TA cloning.
TIA
Hi,
yes it is possible to perform TA cloning first and subclone the fragments later, however, if your PCR product has the same size as another fragment from pGEMT-easy it is not suitable for subcloning.
there are many web resources that provide virtual digests for the sequence that you input i prefer benchling.
Good luck.
https://www.addgene.org/browse/sequence_vdb/2853/
I don't understand Vamsi's point at all.
You should have no problem cloning as you describe. But I usually suggest amplifying first with a proofreader, then adding the tails with taq.
Alexis,
Yes!
As long as your primers have sufficient overlap with your sequence of interest, addition of extra bases for introducing restriction sites should not matter, as long as they do not create some undesirable amplification regions. You can check it using any software. Use only an enzyme with proof-reading ability at this stage to amplify the DNA.
Once you have the right DNA fragment, you have two choices:
1. Performa second reaction using Taq polymerase to add the single base you will need in TA cloning and proceed with your plan.
2. Cut the DNA fragment with unique restrain site(s) you introduced and directly clone the purified DNA fragment into your final vector (also digested and purified with compatible restriction sites).
I see no reason for method 1 when 2 is already more than adequate and does not include use of an enzyme lacking proofreading ability.
Best,
Since the restriction enzymes were not mentioned, my intention was to point to a situation where the enzyme cuts the vector more than once and yields fragments of similar size to the pcr product . that is 2 fragments in the same band
So it would be helpful if a virtual digest is carried out before choosing restriction sites
Vamsi: my guess is that Alexis will have done that already.
Tausif: we used to try the RE digest of the PCR products, but often had problems. And we couldn't tell if one or both of the RE sites in the PCR product had not been digested. So we reverted to TA cloning!
Hi Alexis,
The answer to your question is YES. Your primers designed with restriction enzymes will yield an amplicon with the REs, and extension overhangs if you amplify the DNA with the enzyme you suggested. You will be able to ligate into pGEMT-easy.
You can use Genome Compiler (free) to set up the virtual cloning. I hope this was helpful.
Cheers,
-Antonio
Vamsi:
"Since the restriction enzymes were not mentioned,..."
From the original question:
"Would it be okay to still use the primers I have designed with the restriction sites to clone into pGEM T Easy and then subclone into the reporter vector?"
Geoff:
"Tausif: we used to try the RE digest of the PCR products, but often had problems. And we couldn't tell if one or both of the RE sites in the PCR product had not been digested."
It depends on restriction enzyme site, some require a longer stretch of sequence on both sides of the actual cut site than others. As long as this requirement of size is satisfied, they will get cut satisfactorily. You are correct in that there will be no discernible difference in size on a gel. The system, however, works quite well as long as the design is well-thought.
Yes, we appreciated the end requirement, and designed accordingly. When you don't get the product you want, you start to consider all the possible explanations. Confirming the end digestion is one of the more difficult - self ligation and concatenation after either and both RE, respectively, is doable, but TA cloning was easier for us.
I have a simple tips....I suggest you to add single or double additional bases (C or G) external to the restriction site during primer designing. I don,t know why, sometimes terminal bases are mutated/changed observed after sequencing. Otherwise, performing PCR and TA cloning for long term preservation is very effective for future cloning procedure. Best of luck.
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