overcoming seq. bands in all 4 lanes
dhoffman at spot.Colorado.EDU
Thu May 12 11:05:23 EST 1994
Jonathan Hecht <jhecht at ucsd.edu> writes:
>In article <jcn-060594092912 at 18.104.22.168> Jeff Nichols, jcn at rice.edu
>>Does anyone have suggestions about overcoming bands in all
>>four lanes when using Sequenase? I'm doing dsDNA sequencing.
These stops result from Sequenase's tendency to pause or terminate in regions
with secondary structure too stable for it to read through. There are a couple
of things USB recommends to eliminate these problems: first, you can use the
dITP containing labelling and termination mixes included with the kit, although
they tend to accentuate the pausing in my hands. Second, you can try using the
7-deaza-dGTP containing labelling and termination mixes - in my experience this
works quite well, although when sequencing regions of strong secondary structureit doesn't eliminate all the stops. Finally, there is a protocol from Biotechniques using an incubation with terminal deoxynucleotidyl transferase after the
termination reaction has been run that works quite well. The idea is that
molecules created by pausing of Sequenase probably don't terminate with a ddNTP,
so TdT can extend them; after a 30 minute incubation, most of them are long
enought that they run near the top of the gel with products that are too long for your gel to resolve anyway. Protocol follows:
Prepare a TdT reaction mix (I make 20 ul):
12 ul 1.25 mM dNTPs
4 ul 5X Sequenase reaction buffer
4 ul TdT (17 U/ul; USB)
Mix thoroughly; aliquot 2 ul per tube. Incubate at 37 C for 30 minutes.
Terminate reactions with 4 ul stop solution and run on gel.
If you want the specific reference, let me know, and I can dig it up for you.
This version is a bit different than the one published, but it worked better for
me this way.
>Try sequencing the same region coming from the opposite direction. If
>you don't have an appropriate primer for this it is relatively cheap to
>have one made.
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