I am trying to assemble a vector by Gibson Assembly. I have four fragments (two large and two small) generated by PCR and have to use Gibson to ligate them and get the vector.
After transformation of Chemically Competent TOP10 cells, I had only one colony.
I performed a colony PCR , ran AGE and the band was the right size. I then sent the purified PCR product for sequencing.
The sequence came back with some surprising data. right where the fragment starts, for the first part of it I have 550 bps which NCBI Blast says it aligns with an expression vector I never used and the other half of the fragment is nothing. and again I have the right seq after the fragment.
Can anyone give me suggestions what is happening here and what I need to do next?
The problem with using BLAST to identify the sequence from your construct is that many vectors are not unique as they have been created from portions of other vectors. This may explain the presence of the vector sequence you did not use.
Unless you are using a common vector, it's often better to directly align the sequence results with the expected vector to determine whether they are the same. This can be done with Global Align in NCBI up to 100 kb as well as many other software packages.
What do you mean when you say "the other half of the fragment is nothing"? Is the other half of the fragment producing unreadable sequence? Is the other half of the sequence not producing a hit in BLAST?
An unreadable sequence could be due to distance from the sequencing primer. Sequencing techniques can only reliably sequence a certain distance in bases before a new primer is required to continue to read. Typically the read length could be longer than 550 bases, but there are factors that could contribute to shorter reads. And if Sanger sequencing is being used, the expected limit is only ~900 bases anyway, so it may be that you need another sequencing primer at 550 bases.
If the second portion of the fragment simply doesn't produce a hit is BLAST, should it have? Is this part of a known gene or operon that would be expected to produce a good match? Is this a sequence that was added for some other reason such as multiple cloning sites or a Gibson cloning primer overlap region?
hi Nathan
Thanks for the answer.
when io blast this 550 bp against the expected vector, there is no hit.
also, what I have received from the sequencing company, shows only - - - - - for the other 500-700 bps of fragments and then again after that there is a sequence.
my primer is 200bp upstream of the fragment. I get that 200 +550 and then nothing and then more sequence.
Does this make any sense?
Hi Parisa,
The fact that you obtained only one colony is already worrying. I would not invest too much time in trying to figure out what is going on with your sequencing results but in trying to optimise your Gibson Assembly reaction conditions instead.
First, it would be good to know how large and how short are your fragments. Also, are you using a commercial mix such as NEBuilder HiFi DNA Assembly or a home made mix?
If some fragments are < 200 bp, you should use 5 times more of these in your mix but make sure you do not exceed 0.5 pmols of total insert amount. If some fragments are too short compared to others, I sometimes do an overlap extension PCR step to merge some adjacent fragments (large with short or short with short) prior the Gibson Assembly step. This way, you reduce the imbalance between fragment sizes and the total number of fragments at the same time. Finally, an hour incubation at 50C is necessary when performing complex assemblies ( > 3 fragments) as opposed to the standard 15min. If you are using a commercial mix, it must come with a positive control, make sure you are doing this control in parallel to make sure that nothing is wrong with the mix itself or with the transformation.
Hope this helps.
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