Capillary DNA Sequencing - (Mar/04/2009 )
I am new to this forum, but have heard great feedbacks from many people. Hopefully I will get some help too.....
I am working with a 3130 ABI capillary DNA sequencing machine with a 36 cms capillary. I get good read to about 550 to 600, but beyond that I get huge plateaus and the fret scores are really low. I have spoken to the ABI people and I have got the feedback that I should be happy I got upto 550-600, as they claim only about 500 bases. They are asking me to go for longer capillaries. I wanted if this is really true. Is it not possible to get longer reads with a 36 cms capillary. If so, can you suggest me ways to improve my read length.
I also find that I lose about 30-40 bases of the initial sequence. How can I improve my purification so that I retain the smaller fragments as well. Right now I am following the 125mM EDTA/3M Na-acetate/Ethanol purification method.
Thanking you all to have taken time to read my query and have a nice day.
The longer capillary will likely give you longer reads, but at the cost of a longer run time. We prep'd sequencing reactions with Princeton Separations columns (basically G-10 sepharose, I think) which worked well. But you'll always get bad sequence for the first few dozen bases. Move the primer back if you need to see those bases. A critical parameter if you are trying to optimize sequencing is the DNA concentration. You need enough, but also not too much. You could try serial 2x dilutions of some identical samples on your machine to test the effects and optimize amounts.
Thank you very much for your input. We do not have the separation columns. But as you said, even I see about 30-40 bases lost from the initial read. The DNA concentration, I think may improve the read. I am trying out today, will let you know the results. For a plasmid of about 5 kb, I was using around 250 ng. Now I have done diff concs like 150, 100 and 50ng as template for seq PCR. Keeping my fingers crossed... Thanks once again
phage434 on Mar 5 2009, 10:40 PM said:
Bad News!!! No improvement in the read length !
Have you tried a new capillary? They do go bad after a while. There are (old) threads and papers on cleaning capillaries. I recall proteinase-K and acid washes as the basic trick. But first you should just try a new one. I'm surprised the ABI folks didn't suggest this.
The capillary is not that old, maybe few months, with less than 40 runs
phage434 on Mar 6 2009, 07:42 PM said:
They were right, the sequencing reads are limited by the capillary length. And also kit used. ABI has two kits, Big Dye v1.1 and v3.1.
v1.1 is designed for shorter reads with start after some 30 bp and v3.1 is designed for longer reads (up to 700 bp but depending on capillary and polymer) but with later start, so you loose about 60 bp at the beginning. Capillary needs to be in good condition to get basecall from beginning sequences too (we had recently problems with ours and the reads started more than 50 bp from start wich is almost double the normal distance).
AFAIK you can't have both, early start and long read at the same time. That's the principle of the reaction, if you use more ddNTPs on the beginning, than you will miss them on the end of the sequence.