Expression from a Gateway vector requires two cloning steps:
First, a PCR amplification product is cloned into an entry vector (pENTR or pDONR) using BP clonase.
Secondly, the entry construct is incubated with a destination vector, and recombination is facilitated by LR clonase.
The first step usually involves PCR, the error rate of which is about 1 in 10^3 or so. As such, it is customary to sequence the entirety of whatever is being cloned into the entry vector.
On the other hand, the second step (the LR reaction) does not involve in vitro DNA synthesis. Usually, I just check that the sequence of the vector-insert junction is correct. I typically do not sequence the entirety of the insert a second time, especially if it is a big insert.
What do other people think?
I think with the digestion should be enough. I heard that sometimes the clonase reaction can give concatemers, though it never happened to me
End sequencing should be sufficient.
This depends, of course, also on the number of LR reactions you would like to verify.
I prefer Sanger, but I believe that checking once is enough (personally, I checked after LR - after BP is probably wiser ;) ).
I certainly recommend to check post-BP.
Again, if it is only a small number of destination clones, you can check in-depth, even using full-length sequencing depending on the length of the insert.
If you have hundreds, end sequencing should be sufficient.
Given the average ORF length, this will give you full-length verification for a large fraction and thereby also an idea of your error rate...
I sequenced the amplification product after TA cloning, then the gene was inserted into pENTR2B by digestions and ligations; then LR reaction was done, and the target vector was verified by PCR using the gene specific primers and digestions. sometimes the digestions was not expected. I think to sequence can solve the problem in the latter case.
I always use end sequencing to verify my insert, after first using restriction digest to have the best candidate genes. I never sequence the whole vector but I sequence the insert using the M13F and M13R primer sequence.
As insert length is big, therefore double digestion and end sequencing is sufficient to proceed for next step.
This is a risk/reward question. The odds of getting an internal mutation in a sequence you don't amplify are low, but there is some chance that you'll pick up an error. As others have suggested, you can mitigate the risk by sequencing the junctions.I would make a judgment based on the value of the clone to you. If you're planning to move forward with protein expression, purification, characterization, etc, you might want the reassurance that final full sequencing will give you. On the other hand, you might get lucky.
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