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Forum: Sample Prep / Library Generation 12-18-2017, 02:20 PM
Replies: 0
Views: 582
Posted By kwaraska
Hyperprep on SciClone

We are running the Kapa Hyperprep on SciClone for the first time and are confused by the workbook.

Under post-ligation double sided SPRI what numbers do you enter? We ere thinking 0.4 and then a...
Forum: Sample Prep / Library Generation 07-22-2016, 06:47 AM
Replies: 2
Views: 612
Posted By kwaraska
We always normalize based on total RNA. The...

We always normalize based on total RNA. The transcript population will vary but will be proportional. Keeping the OD of the culture is less important(in my opinion) than starting with equal total...
Forum: Sample Prep / Library Generation 04-24-2014, 11:35 AM
Replies: 1
Views: 1,001
Posted By kwaraska
If you have dbl-stranded cDNA you just use a...

If you have dbl-stranded cDNA you just use a genomic kit from here on out..

You will not get directionality but you still get only transcriptome data
Forum: Illumina/Solexa 04-04-2014, 08:18 AM
Replies: 4
Views: 2,385
Posted By kwaraska
It depends on whether you need both primers or if...

It depends on whether you need both primers or if the "genomic" pool will interfere with your sequencing.

We use 15ul of 100uM primer in 3 ml of HT1 for HiSeq Rapid. We use .6ul of 100uM and...
Forum: Illumina/Solexa 03-21-2014, 12:19 PM
Replies: 2
Views: 1,304
Posted By kwaraska
Nothing specific to running them. It is all in...

Nothing specific to running them. It is all in the way the analysis is done.

The difference is that a stranded(also known as directional) protocol puts one adapter on one side and another adapter...
Forum: Sample Prep / Library Generation 03-13-2014, 06:43 AM
Replies: 1
Views: 1,545
Posted By kwaraska
I asked and it will not work due to the length of...

I asked and it will not work due to the length of genomic.

The only thing that works consistently is old school Ethanol Precipitations.
Forum: RNA Sequencing 03-06-2014, 07:56 AM
Replies: 2
Views: 1,430
Posted By kwaraska
10ng/ul for an RNAseq library is not low-it is in...

10ng/ul for an RNAseq library is not low-it is in fact quite good.

What does your bioanalyzer trace show?
Forum: Sample Prep / Library Generation 03-05-2014, 12:43 PM
Replies: 1
Views: 1,080
Posted By kwaraska
The egel is more hands on. The pippin prep takes...

The egel is more hands on. The pippin prep takes longer but you can set it to do what you need it to and walk away.
Forum: Sample Prep / Library Generation 03-04-2014, 10:33 AM
Replies: 1
Views: 859
Posted By kwaraska
Yes-we run only single sided SPRI. It is mostly...

Yes-we run only single sided SPRI. It is mostly because you cannot control the shearing in ChIP so the sizes may be different. You don't want to lose any sites.
Forum: Illumina/Solexa 02-27-2014, 07:05 AM
Replies: 5
Views: 1,554
Posted By kwaraska
The Fluidigm C1 which is single cell cDNA uses...

The Fluidigm C1 which is single cell cDNA uses 1/4 reactions so I believe it is tied to DNA/cDNA quantity. The only change in the 1/4 reaction is the first PCR step is 10 min instead of 5 min. I...
Forum: Sample Prep / Library Generation 02-27-2014, 06:59 AM
Replies: 1
Views: 2,211
Posted By kwaraska
The lack of reproducibility has more to do with...

The lack of reproducibility has more to do with the BioRuptor itself than with the DNA type.

We switched to a Covaris because of this very issue- it was not reproducible and very difficult to get...
Forum: Sample Prep / Library Generation 02-07-2014, 06:42 AM
Replies: 4
Views: 1,643
Posted By kwaraska
While I know nothing about your organism, I do...

While I know nothing about your organism, I do know that if you denature drosophila RNA the peaks are virtually on top of each other-more like a doublet than two distinct peaks-whereas if you don't...
Forum: Bioinformatics 02-04-2014, 09:15 AM
Replies: 1
Views: 935
Posted By kwaraska
I know from a bad experience that old style SR...

I know from a bad experience that old style SR libraries will not work on the MiSeq.

Only the older style (pre-TruSeq) Paired End work on the Miseq.
Forum: Sample Prep / Library Generation 02-03-2014, 09:27 AM
Replies: 3
Views: 1,366
Posted By kwaraska
Usually if it won;t toggle it is because of an...

Usually if it won;t toggle it is because of an error in one lane.

If you can remove that error, even if it means losing that sample, you can recovere the rest of the run.
Forum: Sample Prep / Library Generation 02-03-2014, 09:18 AM
Replies: 5
Views: 1,285
Posted By kwaraska
I know many kits have very specific maximums and...

I know many kits have very specific maximums and just increasing the volumes doesn't always work.

I know Epicentre has a max of 1ug and over that causes incomplete reduction.

Not familiar with...
Forum: Core Facilities 11-14-2013, 07:49 AM
Replies: 4
Views: 5,470
Posted By kwaraska
You mention you are in Boston. The BPF at...

You mention you are in Boston.

The BPF at Harvard Medical School has a C1.

Here is a link to the website www.genome.med.harvard.edu
Forum: Sample Prep / Library Generation 11-01-2013, 10:14 AM
Replies: 2
Views: 1,980
Posted By kwaraska
This is what I use

http://www.scienceprimer.com/dna-ng-nm-calculator
Forum: Sample Prep / Library Generation 10-30-2013, 06:07 AM
Replies: 9
Views: 4,261
Posted By kwaraska
Are you having them do ribo reduction? This...

Are you having them do ribo reduction?

This trace looks as though they have already done ribosomal reduction in preparation for sequencing. Is that possible?
Forum: Sample Prep / Library Generation 10-09-2013, 11:22 AM
Replies: 2
Views: 1,878
Posted By kwaraska
TruSeq works on cDNA. Just treat it as DNA.

TruSeq works on cDNA. Just treat it as DNA.
Forum: Illumina/Solexa 10-01-2013, 06:58 AM
Replies: 2
Views: 1,882
Posted By kwaraska
Since the P5 and P7 sites are what bind to the...

Since the P5 and P7 sites are what bind to the flow cell, I would assume that would interfere with binding. The P5/P7 sites bind to the oligos on the flow cell and they likely would not bind to it...
Forum: Sample Prep / Library Generation 09-20-2013, 01:25 PM
Replies: 4
Views: 1,880
Posted By kwaraska
It is a valid way to make a library-especially if...

It is a valid way to make a library-especially if you do not have sufficent RNA to put into a protocol. The only disadvatage is that you lose stranded-ness.

Yes-I run the same Covaris program for...
Forum: Core Facilities 09-20-2013, 01:23 PM
Replies: 4
Views: 5,470
Posted By kwaraska
I wish I had advice but we are having the same...

I wish I had advice but we are having the same issue.

The only thing we have discovered is that even though they tell you that you can run the capture up to 3X to increase the capture number on...
Forum: Epigenetics 09-20-2013, 01:20 PM
Replies: 14
Views: 6,622
Posted By kwaraska
New England BioLabs has good kits, small sizes...

New England BioLabs has good kits, small sizes and I don't think you need a methyl-seq kit but you might need a microbiome kit.

I would look into them. Their kits are Illumina sequencer compatible
Forum: Sample Prep / Library Generation 09-17-2013, 01:14 PM
Replies: 9
Views: 3,044
Posted By kwaraska
Generally speaking, nothing larger than about 700...

Generally speaking, nothing larger than about 700 bases will cluster.

I would recommend against a size selection because since the transposase is not completely random, you could be cutting out a...
Forum: Sample Prep / Library Generation 09-17-2013, 01:11 PM
Replies: 1
Views: 1,783
Posted By kwaraska
In the older scheme there was a third indexed...

In the older scheme there was a third indexed primer if I recall.

Illumina has a primer document that includes even back that far. I would post it here, but they do not want us to post it and...
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