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Forum: Illumina/Solexa 03-24-2016, 08:24 AM
Replies: 5
Views: 6,963
Posted By SeqR&D
Hi

Yes, you definitely need methylated adapters...Assuming you are BSC the libraries before adding the adapters, which is likely no.

What is your custom hybridization probe plexity? How many probes?...
Forum: Illumina/Solexa 09-06-2013, 12:34 PM
Replies: 2
Views: 6,581
Posted By SeqR&D
Are your adapters methylated? Do you know that...

Are your adapters methylated? Do you know that you are using the same sequences with the same double stranded/single stranded lengths? What do they look like on a denaturing gel?
Forum: Illumina/Solexa 09-06-2013, 12:24 PM
Replies: 5
Views: 6,963
Posted By SeqR&D
I have perfomed BSC on TruSeq libraries several...

I have perfomed BSC on TruSeq libraries several times and have had no problems other than losses/degradation. I recently tested BSC kits (Promega methyledge, Qiagen Epitect fast, and both the Zymo...
Forum: Illumina/Solexa 10-25-2012, 04:20 PM
Replies: 8
Views: 3,918
Posted By SeqR&D
If you buy a new kit, and run conditions where...

If you buy a new kit, and run conditions where you replace one of the new reagents with one of the old reagents...then you can see which one is the trouble maker. It sounds like you need a new kit...
Forum: Illumina/Solexa 10-23-2012, 02:18 PM
Replies: 4
Views: 2,579
Posted By SeqR&D
why would a company give up a secret that is...

why would a company give up a secret that is making them money, so that you can buy it from someone else? they do give you a way to use your own sequencing primers...what more could you want? :) Can...
Forum: Illumina/Solexa 10-23-2012, 02:06 PM
Replies: 4
Views: 7,249
Posted By SeqR&D
Illumina sells primers with P5/P7 and the indeces...

Illumina sells primers with P5/P7 and the indeces and the adapters. So, to save money, the length of your primers only need to have the locus specific sequence and enough of the index primer...
Forum: Illumina/Solexa 10-23-2012, 02:00 PM
Replies: 8
Views: 3,918
Posted By SeqR&D
I agree...it has to be the enzyme...everything...

I agree...it has to be the enzyme...everything else is buffer.
Forum: Illumina/Solexa 10-23-2012, 01:50 PM
Replies: 24
Views: 6,811
Posted By SeqR&D
adapters

Think about the adapter system. There are either 2 or 4 oligos that make up the Y-adapter system. Next...how does cluster generation happen (how does the FC grab the library?), and what do you need...
Forum: Sample Prep / Library Generation 09-06-2012, 02:59 PM
Replies: 12
Views: 78,730
Posted By SeqR&D
depends on if it is dsDNA (660g) or ssDNA (330g),...

depends on if it is dsDNA (660g) or ssDNA (330g), and assuming you have a volume associated with your DNA. ng/uL * (bp*mol)/660g or 330g * 1/(length of DNA in bp) * 1e^6 uL/1L. this leaves you with...
Forum: General 06-13-2012, 09:48 AM
Replies: 14
Views: 4,811
Posted By SeqR&D
Nothing is impossible

I say not impossible, and not necessarily relyant on the error rate of sequencing, or of amplification. If you can make a sequencing library that is smart enough to overcome these obstacles, you can...
Forum: Illumina/Solexa 05-14-2012, 12:33 PM
Replies: 75
Views: 32,746
Posted By SeqR&D
Genquest

Hi,

My initial thought is...if you are exactly 50% off...maybe you diluted wrong. If you were to see a gradual decrease in concentration, then I would say that your dilutions are getting old....
Forum: Illumina/Solexa 03-10-2012, 07:53 PM
Replies: 22
Views: 10,057
Posted By SeqR&D
If you qPCR a non-ampified sample that has...

If you qPCR a non-ampified sample that has varying amounts of free adapter (with a keenly designed SPRI purification you can greatly minimize this), library elements with one ligated adapter and...
Forum: Illumina/Solexa 03-09-2012, 01:02 PM
Replies: 22
Views: 10,057
Posted By SeqR&D
Neither of those are true. I'm certain that if...

Neither of those are true. I'm certain that if you run a denaturing gel on the adapters, that they will run as 2 bands. And, PPC primers are complimentary to the adapter ends...nothing special.
...
Forum: Illumina/Solexa 03-09-2012, 08:28 AM
Replies: 22
Views: 10,057
Posted By SeqR&D
There is no magic in the PPC. Adapters are not a...

There is no magic in the PPC. Adapters are not a complete "library element"...which is the beauty behind the Y-adapter. You do not have complimentary ends...they are "sticky", and only contain half...
Forum: Illumina/Solexa 03-09-2012, 08:19 AM
Replies: 75
Views: 32,746
Posted By SeqR&D
I agree with Jon Keats on his qPCR thoughts. ...

I agree with Jon Keats on his qPCR thoughts.

1. qPCR is completely dependant on your standards, and your dilution of the standards.
2. qPCR is also dependant on your dilution of your samples.
...
Forum: Sample Prep / Library Generation 06-16-2011, 07:50 AM
Replies: 12
Views: 7,938
Posted By SeqR&D
1. It is recommended to use freshly prepared 80%...

1. It is recommended to use freshly prepared 80% EtOH.
2. 15 min incubation to bind DNA with multiple vortexing steps during this time period...~10sec per vortex should be fine...just mix things up....
Forum: Illumina/Solexa 09-20-2010, 02:44 PM
Replies: 22
Views: 10,057
Posted By SeqR&D
I wouldn't think so. The most difficult part here...

I wouldn't think so. The most difficult part here is aligning your qPCR results to your cluster #. After that, your cluster # should not change dramatically enough to have to redo the titration. If...
Forum: Sample Prep / Library Generation 09-16-2010, 11:54 AM
Replies: 4
Views: 2,897
Posted By SeqR&D
Okay...I get it. Yes, I do this PCR...both...

Okay...I get it.

Yes, I do this PCR...both to add the correct sequence for sequencing, and to ensure that you have library with correctly orientated adapters. I generally do 14 cycles, but this...
Forum: Sample Prep / Library Generation 09-16-2010, 10:34 AM
Replies: 4
Views: 2,897
Posted By SeqR&D
Hey James, What is PCR enrichment? I have...

Hey James,

What is PCR enrichment? I have made many PE libraries and do not run into too many issues. I follow the illumina protocol. Have you tried making these libraries yet? How much DNA are...
Forum: Sample Prep / Library Generation 09-16-2010, 10:23 AM
Replies: 3
Views: 4,207
Posted By SeqR&D
I would double check your primers compared to...

I would double check your primers compared to what you are putting on as adapters. If you have material, but you cannot amplify it...there is a simple reason.

1. Primers do not match/hybridize to...
Forum: Sample Prep / Library Generation 09-15-2010, 04:01 PM
Replies: 12
Views: 78,730
Posted By SeqR&D
Okay, I'm not sure that anyone answered your...

Okay, I'm not sure that anyone answered your question.

500ng/uL * 1x10^6 uL/L * bp mol/660g * 1/350bp = 2,164.5nM

ssDNA is 330g/bp mol

Pico Green is a great way to quantify, if you have the...
Forum: Sample Prep / Library Generation 09-15-2010, 03:54 PM
Replies: 12
Views: 7,938
Posted By SeqR&D
Just watch your elution volume with the Ampure...

Just watch your elution volume with the Ampure beads. If you add 90uL of beads, your min amount to elute is ~30uL for best results...you can stretch with 20uL.
Forum: Illumina/Solexa 09-15-2010, 02:41 PM
Replies: 22
Views: 10,057
Posted By SeqR&D
This is how we did it: 1. We wasted a FC ...

This is how we did it:

1. We wasted a FC
2. Amplified (~14 cycles) gel purified/size selected library (in the size range that you use to sequence)
3. Purified the PCR products
4. Nanodrop...
Forum: Illumina/Solexa 09-15-2010, 02:23 PM
Replies: 16
Views: 8,527
Posted By SeqR&D
Adapter to library ratio is around 10:1

Adapter to library ratio is around 10:1
Forum: Introductions 09-15-2010, 02:17 PM
Replies: 0
Views: 862
Posted By SeqR&D
Thumbs up Hello All

First time on this site, so looking forward to learning and maybe lending a hand where I can. I am an RA in San Diego, have done work on the GAs mostly and I dabble in whatever direction the tide is...
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