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You shouldn't need to do it in two steps, I have used tailed primers of over 100 bp in length successfully.
Assuming that you are wanting to then clone the tagged gene your primers will be something like : 3-6bp RESITE 3bp ATG HATAG 3bp FLAGTAG 3-6bp GENEOFINTEREST (with or without the ATG on the gene of interest, approximately 20 bp of the gene). You can also add a consensus Kozak or Shine-Delgarno sequence upstream of the start codon to enhance expression of the gene in eukaryotic and bacterial cells respectively.
#145657 Tagging a gene with HA and FLAG using PCR
Posted
on 21 November 2012 - 12:32 PM
#145476 How to join two sequencing files taken form forward and reverse primer
Posted
on 18 November 2012 - 09:37 AM
Yes, you don't need to reverse anything for BLAST2seq, it will find the sequence even in reverse orientation. ClustalW AFAIK not.
(but any sequence viewing software like Chromas or FinchTV should be able to reverse the whole plot including sequence, this is generaly not only good for searching within sequence, but also if you want to print sequencing plot from reverse primer in forward orientation)
(but any sequence viewing software like Chromas or FinchTV should be able to reverse the whole plot including sequence, this is generaly not only good for searching within sequence, but also if you want to print sequencing plot from reverse primer in forward orientation)
#145470 How to join two sequencing files taken form forward and reverse primer
Posted
on 18 November 2012 - 07:51 AM
In our lab we use Sequencher for aligning our sequences because our university has a site license for it, but you can align the sequences without purchasing software by putting your non-coding strand in a reverse-complement calculator (like http://www.bioinform...s/rev_comp.html). Then you can use the output in any sequence alignment site you want (BLAST or ClustalW or whatever, although they may handle converting to reverse complement themselves; I didn't check), or you can just look at your forward sequence, find a series of 8 residues or so and search for them in the second sequence. Once you know how they match up, you just have to copy and paste the extra sequence from your reverse primer reaction onto the end of your forward sequence.
Does that make sense? I'm sure there are good sequence alignment sites out there that would do this for you, but I'm not familiar with them.
Does that make sense? I'm sure there are good sequence alignment sites out there that would do this for you, but I'm not familiar with them.
#21189 Creating empty vector by self-ligation
Posted
on 07 April 2009 - 04:17 AM
perneseblue, on Apr 7 2009, 12:16 PM, said:
Not I and Sal I are not compatible. The ligation will not work. You will have to revise the strategy. Ligating an oligo containing ends which are compatible to SalI and NotI would solve the problem.
TC does raise a point. Is creation of a blank plasmid really necessary?
TC does raise a point. Is creation of a blank plasmid really necessary?
You could also fill your sticky ends with Klenow, and then follow to ligate a blunt ended vector, which should work quite nicelly.
I'm assuming you want an empty vector for some transfection controls, if not... I add to TC and perneseblue, is it really necessary?
