Hi Jodie,
Since you seem to get better results with the PCR product in a vector, I
suspect that the vector is helping to minimize the formation of secondary
structure.
For DTS (difficult-to-sequence) templates, I generally don't include any
additional reagents such as DMSO or betaine. However, I do make several other
adjustments which typically break through DTS motifs in the templates:
1) Denaturation temperature is 98C for both the 'hot start' and the cycling
steps. For the 'hot start', I generally use only 2 minutes, but you might need
longer (perhaps up to 5 minutes) if your product is creating really strong
secondary structure.
2) For long templates (>900 nt), I bump the number of cycles from 25 to 35;
however, your short product (<400 nt) probably won't benefit from more cycles.
3) For standard cycling, I use only 0.5 ul of BigDye (with 3.5 ul of 2.5X
Sequencing Buffer) in a 10 ul reaction; however, for DTS samples, I typically
use 1.0 ul of BigDye (with 3.0 ul of 2.5X Sequencing Buffer). The standard ABI
reaction would call for 4 ul of BigDye in a 10 ul reaction (8 ul in a 20 ul
reaction).
For my normal cycling, I've gone to using 95C (10 s), 50C (5 s), 55C (5 s), 60C
(just 2 min -- not the standard 4 min). I tested down to 1 minute extension
times, and even that usually worked... but not always. When I am working with
DTS templates, I do bump the extension time to 2.5 minutes, but these are
typically long templates. Given that your product is so short, I would suggest
minimizing the extension time as much as possible to minimize the formation of
secondary structure... perhaps even dropping to 1.5 minutes. I also tested
increasing the extension temperature in the hopes of minimizes secondary
structure; however, above 62C, signal strength dropped dramatically... so, 60C
seems to be the upper limit for good polymerase activity.
Annealing Temperature: I have found very few primers that require staying at
50C for the full 10 s. However, recently, I dealt with some extremely high A/T
content samples (and primers), and actually had to drop the annealing
temperature to 45C for 45 s (not positive we needed 45 s, but that was the
client's PCR annealing temperatures, so we went with it). If your primers have
a high annealing temperature, consider bumping the annealing temperature...
possibly even to the point of 2-stage cycling (denature at 98C; anneal/extend
at 60C)... to help minimize secondary structure formation.
To summarize, I would suggest you try the following:
1) Skip the betaine.
2) Increase all denaturation temperatures to 98C.
3) If your BigDye input is very low compared to the ABI protocol, try doubling
the amount of BigDye (similar to above).
4) Shorten the annealing time and consider increasing the annealing temperature
based upon the Tm of the primer.
5) Shorten the extension time to 2 minutes (possibly even just 1.5 minutes).
Rest of post
Scott
-----Original Message-----
From: ABRF Discussion Forum <email obscured>> On Behalf Of jfrankl1
Sent: Thursday, December 23, 2021 7:35 AM
To: <email obscured>
Subject: Re: [ABRF Discussion Forum] Sanger Sequencing odd data......
Thanks for replying Scott.
The cycling parameters are
95 degrees for 3:30
Three Step: 95 for :15 then 50 for :15 and then 60 for 4:00
We have sequenced the gel purified PCR products and we see what I described
above. We have also tried sequencing the PCR product placed in a vector and
the QC drops off after the second repeat even though we are using our "RNAi"
mixture which contains betaine. We typically will use this if somebody is
expecting any type of repeat.
On the gel the band size appears to be correct.
Jodie
――
View topic
https://nam04.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F7yNGP1d7K7ZXgjefIPH74d&data=04%7C01%7Csherke%40lsu.edu%7C3c38707cf9c148918b3e08d9c61906a2%7C2d4dad3f50ae47d983a09ae2b1f466f8%7C0%7C0%7C637758633056214093%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=7%2B0L5BH7vE4KrEfV1elmpLbsq0moG%2BEWQHHJw2bx1Gk%3D&reserved=0
Leave group <email obscured>?Subject=Unsubscribe