ABI BigDye Xterminator problems.

CFergie

New member
Nov 16, 2015
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#1

We've been using ABI BigDye Xterminator to clean up our sequencing PCR rxns (spa & 16S) and are wondering if could be to blame for our high rate of sequencing failures. sometimes as many as 1/3 of the 96 well plate fail. We use the ABI 3130 instrument. any other possibilities? Thanks so much!
 

renatohm

New member
Nov 16, 2015
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#2
Well, 1st you must purify your PCR. I suggest you using a volume of 50+ µL of PCR. QIAEX works fine (no need to run the agarose mehtod if you have only 1 band). Trying to analyze the DNA on spectrophotometer is hard because of stuff in the purification kit, so have faith and use 3+ µL of the purified PCR as template for sequencing. We currently make sequencing using 2 µL of BigDye, 2 µL of 5x buffer (save money), 1 µL of 10 pmol primer, 3-5 µL of template (purified PCR) or 1-3 µL (miniprep), Milli-Q water to 10 µL. After cycle sequencing using standard AB protocol, we add 45 µL of SAM solution, vortex the golden resin for 10 sec., pipette 10 µL of it using a wide-bore tip. Pipette no more than a row before vortexing the resin again. Vortex the plate for 30 min, centrifuge @ 1200 xg for 2 minutes and it is done. Put the septa in the plate, download and install the specific 3130 run modules for Xterminator at the AB site and go for it. Xterminator for us works like a charm using this protocol, consistently giving nice 900+ bases with high quality, losing very few samples, 2 in 60, for example.
 

ahsan81

New member
Nov 16, 2015
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#3
Dear Sir,

we are some time facing the problem with XTerminator solution by ABI Big Dye XTerminator purification kit. after some time Xterminator solution become so thick that it cant be used by mean of pipette (it might dry / something else). please tell me any one who face this problem & overcome this issue.