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Forum: Illumina/Solexa 11-19-2014, 08:09 AM
Replies: 18
Views: 6,255
Posted By ZAAB
Seems to me that one of the biggest 'deterrents'...

Seems to me that one of the biggest 'deterrents' of contamination between samples within one prep, between preps across time, etc is to use enough template DNA so that any contamination won't be...
Forum: Illumina/Solexa 05-08-2013, 06:35 AM
Replies: 17
Views: 8,478
Posted By ZAAB
Agreed

We've run quite a few 'expired' kits...

Never had a problem at all...Data Look great.

We cut anything with a Qscore <30, though...but that yield is still ~85% of total reads.

SInce the...
Forum: Illumina/Solexa 10-30-2012, 06:52 AM
Replies: 43
Views: 25,569
Posted By ZAAB
If you're talking about the actual oligos on the...

If you're talking about the actual oligos on the lawn when you say the P5/P7 region, NO, we do not design the sequencing primers to anneal in that region...in our system there is no need for that...
Forum: Illumina/Solexa 10-17-2012, 05:18 AM
Replies: 43
Views: 25,569
Posted By ZAAB
Our custom primers are 75-80deg Tm

We use a plethora of custom sequencing primers for the MiSEQ, and we shoot for 75-80 degrees Tm...I have not tested lower Tm primers, not worth the risk. We just pay for the longer oligo synthesis,...
Forum: Illumina/Solexa 04-18-2012, 05:14 AM
Replies: 9
Views: 9,445
Posted By ZAAB
When we build our own adapters, we perform this...

When we build our own adapters, we perform this using standard desalted primers as well.
We add about half of the illumina adapter seq in a primary PCR (we're sequencing a PCR product whose 5' and...
Forum: Illumina/Solexa 04-18-2012, 03:39 AM
Replies: 9
Views: 9,445
Posted By ZAAB
WE've been using standard desalted custom...

WE've been using standard desalted custom sequencing primers for over a year now, and have had excellent results...
Just remember to get the Tm up to ~70 degrees, so if this requires longer primers,...
Forum: Sample Prep / Library Generation 02-06-2012, 07:57 AM
Replies: 3
Views: 3,137
Posted By ZAAB
My PCR primers for generating the construct...

My PCR primers for generating the construct BEFORE illumina adapter ligation are just regular primers.
I used the Illumina DNA sample prep kit, so the primers were Illumina, and hence, should have...
Forum: Sample Prep / Library Generation 02-06-2012, 06:05 AM
Replies: 3
Views: 3,137
Posted By ZAAB
50+% of my HiSEQ reads are 3' primer (custom primer used)

Hi All:
We've run 7 lanes of Illumina sequencing with the HiSEQ.
We're sequencing the middle of a PCR product...
We're using a custom sequencing primer to avoid the common 5' end of the PCR...
Forum: Illumina/Solexa 06-20-2011, 04:20 AM
Replies: 9
Views: 9,445
Posted By ZAAB
Late reply: Illumina states "HPLC is what we...

Late reply:
Illumina states "HPLC is what we use in the lab".
In the trenches, the guys in my core facility says desalted is fine.
Just make sure your Tm is ~74 degrees, and calculate using...
Forum: Sample Prep / Library Generation 06-15-2011, 09:05 AM
Replies: 4
Views: 4,635
Posted By ZAAB
I've taken this library prep through to...

I've taken this library prep through to completion with three different samples at this point, and I see that the ligation TIME is the screwy part...however I like the idea of decreasing adapter...
Forum: Sample Prep / Library Generation 06-07-2011, 07:40 PM
Replies: 4
Views: 4,635
Posted By ZAAB
Hey: Thanks for the reply on my separate...

Hey:
Thanks for the reply on my separate post...
http://seqanswers.com/forums/showthread.php?p=43479#post43479

I'm just going to clone that 550nt beast and let illumina (and everyone here) know...
Forum: Sample Prep / Library Generation 06-06-2011, 01:31 PM
Replies: 6
Views: 3,548
Posted By ZAAB
Nah...SYBR or EtBr were both in the gel prior to...

Nah...SYBR or EtBr were both in the gel prior to running it.

15-30minute ligations...OK. thanks. And yes, I agree that some degree of ligation occurred since there's an increase in the amount of...
Forum: Sample Prep / Library Generation 06-06-2011, 12:48 PM
Replies: 6
Views: 3,548
Posted By ZAAB
reply

Thanks for the reply.
Adapter ligation step, per the manual, is 10 minutes...(seems WAY too short for me).
Recommendations? I was thinking o/n (like I do for any plasmid constructs).

I've run...
Forum: Sample Prep / Library Generation 06-06-2011, 08:35 AM
Replies: 6
Views: 3,548
Posted By ZAAB
Library PCR enrichment questions (with image!)

Hi All:
I'm preparing libraries (five of them)...all are PCR products to begin with...so size is very uniform, and I avoided the fragmentation and end repair. I picked up at the A-tailing, moved...
Forum: Sample Prep / Library Generation 06-03-2011, 06:12 AM
Replies: 4
Views: 2,166
Posted By ZAAB
I guess I was worried about overloading the...

I guess I was worried about overloading the ligation reaction with insert. I am never convinced that all DNA is carried forward when there is a wash/Ampure step.
Thanks for all replies.

I...
Forum: Sample Prep / Library Generation 06-03-2011, 04:03 AM
Replies: 4
Views: 2,166
Posted By ZAAB
Exactly! Yes, I am using Taq, and Yes, I do not...

Exactly! Yes, I am using Taq, and Yes, I do not need to shear...
Just interested in the amount of DNA from a 1ug shearing 'reaction'..what amount is of the correct size...80%? I'll have 100%, so I...
Forum: Sample Prep / Library Generation 06-02-2011, 12:39 PM
Replies: 4
Views: 2,166
Posted By ZAAB
150nt insert size, generating libraries, question!

Hi All:
I'm preparing libraries for the HiSeq...
We're generating a library of variants, and the whole construct is 143nt long...with 30 nt in the middle that are variable (lots of variants!).
...
Forum: Illumina/Solexa 05-03-2011, 07:00 AM
Replies: 0
Views: 2,573
Posted By ZAAB
DIY illumina adapters

Hi All:
Trying to determine whether I can get away with making my own adapters for the HiSEQ 2000.
I've got a library of small DNA (125nt) fragments, and would like to sample the diversity of...
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