Hi everyone,
I'm sequencing a gene post site directed mutagenesis and so far I'm getting horrible results with poor coverage. For the same gene I sequence twice using the T7F and T7R primers. I get coverage of ~160 BPs using the T7F primer and a lot less using the T7R primers.
My insert is 900 BP and my plasmid is 6 KB. I don't preform clean up prior to sequencing but I usually use quite a high concentration of DNA.
Can anyone point me in the right direction?
Thanks for your time
Harry
Use abt 50ng of plasmid DNA for sequencing with your 2ul of T7 primers (1.5-1.75 uM concentration ) for sequencing in 3770xl analyser.
Hi,
We typically do mini-prep and sequence. I recently did sequencing of direct PCR mix. It works very well.
Design new primers, mention if its PCR mix, cleanup, plasmid to the company,
they modify protocols to sequence accordingly.
We send 500ng plasmid for sequencing. If its GC rich, mention that in sequencing form.
Good luck..
can you post a .ABI file for inspection please? Also the amount of dna that you put in the sequencing reaction
- Badges
- Science method