Paired end library, 3kb woes

[ringo1230]

I have a question.
There is only the amount of 10ng DNA after reaction Plasmid-Safe ATP-Dependent DNase. I'm worried about it is that the experiment failed. Is it that the amount of DNA remaining everybody how much?

[MissDNA]

Oh and as I mentioned before, when I see the peak after nebulization is too low, I proceed with 2 library amplication reaction and combine both in the last purification column step.

[MissDNA]

It is the purified/sizing slected library. I don´t run any chip or gel after the amplification reaction. As I told you before, I follow the protocol as is. The only modification I do is: after shrearing the DNA, I don´t do the ampure cleanup instead I concentrate my sample in a QIAquick column.

I use the concentration value and avg size I get from the Pico Chip, and throw in some excel spreedsheet I got from Roche years ago. That gives me the number of molec. and the values to make a 10e08 dilution to further dilute and use in the emPCR. Some of the libraries I have sequenced had barely 10e08 molec, so we made emPCR straight out of it and sequencing worked fine.

The 3kb PE library we sequenced yesterday yield great results, so great I am scared there is something wrong. It was a poor library. I am super curious to see the assembly.

[Dun]

Two questions, Miss:

The RNA trace you showed in other post, is it purified/sizing selected sample or straightly out from amplification reaction?

What is the amount of your final library?

THX a lot.

[Dun]

Yes, it is amplified library by PCR.

[MissDNA]

When you say PCR product, do you mean the amplified library?

Your trace shows a lot of short fragments. My barely there peaks start raising at about 300 bp and end at around 1000 bp.

I would like to see your final library and know the results of your sequencing, especially if the RLMID worked.

[Dun]

My trace after Covaris 500bp fragmentation looks very similar to your nebulized ones.

[Dun]

Many thx. I will check out that post carefully, seems it's a common problem.
I don't have HS report with me right now, I can show a pic of PCR product which is done by DNA 7500 chip.

I will throw my PE library in anyway later this month.

[MissDNA]

My traces are on this thread: http://seqanswers.com/forums/showthread.php?t=16183

[MissDNA]

I see. I have never tried that and I am not really sure that would work.
I have posted in some other thread, traces after nebulization where there is an almost flat line and then the final library in the expected size range. I´ve never seen a nice peak after nebulization.
Do you have the trace of an HS chip?
It would be actually interesting to know if your libraries would sequence well.

[Dun]

thx MissDNA

So it's OK to have flat line after circularization?

The story after that is a little complicated. since we are trying to put RLMID adaptors and use rapid library protocol, so the fragments I was mentioned are dsDNA.
I am wondering how does the DNA trace look like right after PCR in original assay.

[MissDNA]

Why don´t you follow the protocol, using nebulization, carrier DNA and 15 cycles? Even though some libraries to not look perfect on the RNA chip, they tend to sequence well. I have prepared and sequenced over 30 3kb PE libraries, which good results.

We sequenced another 3kb PE library today. I will see the results tomorrow morning. The library was definitely not the greatest.

Could you post the agilent trace from your libraries?

[Dun]

Same problem here. During three rounds of trying, I NEVER see any peak after circularization, nor after 500bp fragmentation (I use Covaris). Absolutely no peak.
BTW: I didn't use their 'carrier DNA'

Basically, I didn't see anything after circularzation until PCR, which contains 25 cycles and yields some fragments ranging from 100~700bp. Although I've made 3 PE libraries, I am just simply not trust them, so I didn't put them into emPCR.

I desperately need some direction.
How do you guys do QC for circularzation and final PE library? THX

[MissDNA]

I´ve used different lots of Cre recombinase and I always see an almost flat line after nebulization. However, as I stated before I end up with library. I never checked its stability and since it takes forever to get enzymes in Brazil is hard to keep changing lots.
I sequenced the libraries I made last week. For one of them we got almost 700k reads and median read legth of 418, which is excellent for PE. The other library was bad (barely 10e08 molec./ul) but since we were in a hurry we sequenced anyway. We got about 400k reads with median lenght of 382, most of the reads failed in the mix+dot.

[newmoon]

You should check Cre recombinase activity using the control in the kit. It's not a stable enzyme.