sequencing with large plasmids - (Oct/05/2005 )
Hi,
I have cloned my constructs into pCAMBIA binary vectors. But whenever i give the plasmid DNA for sequencing(we get it done on commercial basis) we never get legible readable sequences. But when we give smaller plasmids like T/A cloning vector or pUC18, we do get good sequences.
Has any one faced such problems?If so please give your feedback in this regard.
thanks in advance
yamini
How big is your construct??
we routinely sequence pGEMT clones with inserts and use 200ng to sequence, for larger vectors (such as BACs or YACs) you would require more sample to obtain similar copy numbers with a smaller plasmid.
when we sequence BACS we use 1ug and perform upto 80 cycles for sequencing, there are specific protocols for large vectors and maybe you should inform the sequencing cervice of this.
Nick
we routinely sequence pGEMT clones with inserts and use 200ng to sequence, for larger vectors (such as BACs or YACs) you would require more sample to obtain similar copy numbers with a smaller plasmid.
when we sequence BACS we use 1ug and perform upto 80 cycles for sequencing, there are specific protocols for large vectors and maybe you should inform the sequencing cervice of this.
Nick
Thanks for the details. My vector is 9kb and the insert(construct) is 1.5kb. Actually few clones have the potential of forming secondary structure because i have cloned the gene in the sense and antisense orientation with a stuffer DNA in between. May be this could be a problem while sequencing but there might be some way out. isn't it?
But i am facing problems with other clones too where there may not be such problems.
thanks
yamini
You could be using too little of DNA for your sequencing, considering your insert to vector bp ratio, however keep in mind when adding large amounts of your DNA into a sequencing reaction you are also adding large amount of any salts, etc., that are on your DNA. I have found that after precipitation, if I wash my DNA 2 times with 70% EtOH, and resuspend in pure H20, I get longer and stronger peaked sequencing.....but hey, maybe it's just me.
10.5 kb vector + insert is not large (I've sequenced from plasmids three times as large). If you're having difficulty sequencing, it's not because your plasmid is too large...
I would look elsewhere -- purity of DNA, amount of template, primer to template ratio, primer design...