These are the results of my BAC tests:
2ug DNA (standard prep with Qiagen midi/maxi)
30pmol primer
16ul BigDye Premix
QS dH20 to a total of 40ul
95C for 5 min, the cycle 100 times: 95C for 30 sec then ramp to
4C
55C for 20 sec
60 for 4 min
Purification works better in an individual spin column versus a plate. Be
sure to bring up in LESS loading buffer (may want to bring up in 4ul and
load 2ul) OR you could load all. I have loaded all, but they can blow out
the lanes next to them, so I've loaded every other lane (on the 373aXL).
Note that some primers give better sequence than others. Also, If you don't
have enough DNA, you'll get some data, but all the T's will be dropped
out--don't know why.
Hope this works for you as well as it does for us (650-700bp)
Dina Leviten
ICOS, Corp.
dleviten@icos.com
> -----Original Message-----
> From: Margarita Almeida [SMTP:malmeida@molbio.med.miami.edu]
> Sent: Wednesday, July 28, 1999 9:52 AM
> To: Recipients of ABRF List
> Subject: DNAseq
>
> I work in a DNA core facility and I am having problems sequencing large
> templates (BAC,PAC,COS & P1). Since the samples received are prepped in a
> variety of way, I was wondering if anyone out there could suggest any
> protocols that I could suggest to my customers. I would also like to know
> if
> anyone has any suggestions for the reaction set up and/or post reaction
> purification. I am using an ABI 373a with XL upgrade and the "Big Dye"
> Terminator kit. Thanks.
>
> Margarita Almeida