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Old 07-10-2012, 11:48 AM   #1
Dynamac
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Location: Cincinnati, OH

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Default Sequencing Short PCR Products

We have no experience in Sanger sequencing of small products, 70-100 bp. So far, we've figured out that we'll need BigDye v1.1, a new spectral calibration, maybe a different POP version, and likely a new way to purify the PCR products because Qiagen columns cut off around 100 bp. We'd like to do the cleanup in plate format. In short, we need advice, big time!

Any suggestions, comments, resources out there? Perhaps it is best to just outsource?

Thanks.

Barry
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Old 07-16-2012, 03:23 PM   #2
rzero
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Hi, we typically sequence 200-300. Using ExoSAP and EtOH precipitation do work well almost equivalent in quality of the final signal as a column-based method. I recommend using v1.1 (which can be used at 8-fold dilution) and POP6 polymer. You could try the below cond to start with:


Polymer 3130 POP-6 Polymer
Capillary Array 16 x 36 cm
Oven Temperature 60 C
DC Injection Time 7 sec (increase may be needed for smaller quantity of amplicon)
DC Run Time 1800 sec
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Old 07-17-2012, 03:44 AM   #3
Dynamac
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Thanks. We'll probably try the ExoSAP. But I'm afraid we're stuck with POP-7 because the 3730xl doesn't support POP-6.
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Old 07-23-2012, 02:24 AM   #4
MrGuy
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we use pop7. Just run a slower run module and BD v1.1. There's another dye kit that just came out that will sequence closer to the primer, but requires a bit more thinking about the approach (can't recall exactly). You don't really need to clean the PCR product, just dilute with water (1:10 or 1:100) or do exosap (or exo and sap).

I use xTerminator for cleanup instead of EtOH (better reproducibility, higher cost for xterminator). Run a low volume (10uL or less) sanger reaction to save on costs.

Another easy trick is to tail the primer so you can sequence through the primer first (ie, low quality) then go to the sequence fo interest.
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Old 07-23-2012, 10:27 AM   #5
Dynamac
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Thanks. I'll investigate these ideas. Too bad we can't do this with the 454 platform. I know for a fact that it LOVES small fragments.
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