Tapestation shows additional bands

[Tamar]

Hello everyone,

I have been working according to the Joung et al 2017 paper and wanted to ask whether anyone has gotten additional unrelated bands on tapestation? 

I pooled the pcr reactions from my amplified library and purified them on Zymo columns. Then I ran 2 micrograms from each sample on an agarose gel at low voltage so as not to heat or denature the dna. I saw a clear band at slightly below 300bp (my size should be 260-270 bp), cut this from the gel and purified the dna from the gel making sure not to heat the sample. But when I measured this sample in Tapestation I saw a peak at 280bp and a peak at 500bp. Does anyone knoe what this means? I saw previous threads that said that this is ssDNA that denatured and reconnected creating secondary structures that run larger. Yet if so, how can I quantify this in qubit to make sure that I put equal amounts of each sample in the pool I send for NGS?

Thanks!!

[Donghee Lee]

Hi, Tamar

You don't need to put equal amounts of each sample, because you can specify each group by barcodes in reverse primers.

MaGeck algorithm will normalize your data. Just believe quibit data as it shows. Don't worry.

Best,

Donghee Lee

2019년 7월 21일 일요일 오후 9시 43분 4초 UTC+9, Tamar 님의 말:

[Tamar]

Thanks very much for your answer. But dont you have to put equal amounts from each sample in order to compare enrichment between the different barcoded samples?

[Donghee Lee]

Tamar,

Maybe you prefer MiSeq for screening. I usually do HiSeq (I do not mix my samples), because it's cheaper in my case.

and I produce equal amounts of reads for each sample.

However it's not exactly same, so I normalize read count of each sample with total read count with MaGeck software.

So, I pcr the same amount of gDNA, and then I do not qubit my NGS library sample. 

I just make enough amount of NGS sample & read the same amount for each sample.

Protocol may vary by a case.

Best, 

Donghee Lee

2019년 7월 22일 월요일 오후 6시 37분 39초 UTC+9, Tamar 님의 말:

[Julia Joung]

Hi Tamar,

It's possible that you overloaded your agarose gel, and did not successfully extract your target band cleanly. Of course, this depends on your well size, but it might be worth looking into. Typically, the higher molecular weight band is much lower in abundance and does not cluster as well on the MiSeq because it is much larger, so it is not usually a concern.

Best,

Julia

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[Tamar]

Thanks very much for your answers!

On Tuesday, 23 July 2019 16:53:27 UTC+3, Julia Joung wrote:

Hi Tamar,

It's possible that you overloaded your agarose gel, and did not successfully extract your target band cleanly. Of course, this depends on your well size, but it might be worth looking into. Typically, the higher molecular weight band is much lower in abundance and does not cluster as well on the MiSeq because it is much larger, so it is not usually a concern.

Best,

Julia

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