[DiatomsRule]

Hi! I am having trouble with circularization efficiency as well. You mentioned maybe trying increasing the incubation time, have you tried this yet? Does anyone know what the expected yield percentage should be? Thanks!
Rhonda

I think there has been discussion about this already, but the full scale reaction performs different than the trial.

You need more cycles because the beads inhibit the reaction slightly. You can also increase the reaction volume to overcome this.

[koala]

Good news for the trial PCR. I got the library with 10 cycles...
The problem now is that large scale PCR failed!!
I think that micro columns purification is not a very good step. Isn't so unthinkable that beads seal the columns??

[koala]

Hi Phillip,
thanks for your answer.
I usually look at how many molecules of DNA I could have at each step. The problem is that, often, the samples measured with different instruments (nanodrop, Qubit, agilent) give such different amount of DNA (and this is quite strange). So, I usually take into account Qubit measures and then a confirmation on Agilent bioanalyzer.

For the last library, the measure before circularization was of 800-900 ng of DNA (agilent and Qubit), after, as I said, was of about 17 ng. I've to say that this last measure was made only on the Qubit. As you can argue the efficiency of circularization is very very very low.

I'd like to incease time of MP-ligation incubation and time of circularization, but I've no idea about how long could I try.

Another question is about columns purification after circularization. Why should I use columns instead of XP purification as in other steps? Is due to stopping DNA pol I activity? On my own experience columns produce much more loss of DNA than XP ampure beads.

Finally, a world about relationship with AB assistance. We are in a strictly contact with the italian support and this MP library problem is very well known at AB. We have already tried different solution, but this low efficiency in the library is still unknown...

Best regards
Chiara

[pmiguel]

hello everybody.
I'm trying to work out a MP Solid library of 3 kb. I've some problem with circularization efficiency... I'm not sure to go on the protocol because there is no definition about quantity. I mean, the last measure is before circularization. I had 1ug of DNA; after circularization and columns purification only 17 ng (tot). I've read from some posts that someone has no peaks in agilent, but the trial pcr was ok. What does it means was ok?... How many cycles do you think are good?
In past I had some libraries... but with 16-18 cycles... I' not sure this is a good job, because of repeatitiveness in sequencing.

Thanks all

I would look at it from a view point of how many molecules of usable DNA I have at each step. Rule of thumb: 1 ng = 1 billion (10^9) 1 kb fragments. If your "17 ng" amount is correct, then you would have 17/3 billion molecules. That could work. Circularization tends to be the least efficient step.

The problem is that if you measured that 17 ng by UV spectrophotometry, then the figure is likely not correct.

I would suggest looking at the library on a High Sensitivity DNA bioanalyzer chip prior to PCR amplification. If it is not visible there, it may not be worth continuing. Because even 10 pg/ul would probably be visible on a chip of this type. Since the fragments are less than 0.5 kb at this point, then a flat bioanalyzer trace at this point would suggest that you have less than 20 million fragments/ul. Since not all of them will have adapters attached, this is unlikely to be a successful experiment. Depending on what you want the data for...

3 kb MP libraries should work fairly well. Do you have an Applied Biosystems Field Application Specialist? If so, you might want to ask them to visit you and do the protocol with you.

--
Phillip

[koala]

hello everybody.
I'm trying to work out a MP Solid library of 3 kb. I've some problem with circularization efficiency... I'm not sure to go on the protocol because there is no definition about quantity. I mean, the last measure is before circularization. I had 1ug of DNA; after circularization and columns purification only 17 ng (tot). I've read from some posts that someone has no peaks in agilent, but the trial pcr was ok. What does it means was ok?... How many cycles do you think are good?
In past I had some libraries... but with 16-18 cycles... I' not sure this is a good job, because of repeatitiveness in sequencing.

Thanks all

[pmiguel]

Why increase the amplicon insert size? SOLiD 5500 only does 60 nt reads off each end. The distance of the mate ends from each other is controlled by the original fragment size prior to circularization. Not the final insert size.

Constructing mate end libraries from large fragments (>5 kb) is difficult. Partially this is because your molar concentration of insert drops 10x when you go from 1 kb initial fragment size to 10 kb initial fragment size. But I think this is only a small part of the issue. The large part is that the distance of the the ends from each other is up to 10x longer for a 10 kb fragment than a 1 kb fragment. If the ends do not meet, they cannot anneal.

Seems like back in the day we used to load up ligations with PEG as a "crowding agent" but dilute the reaction up to 500 ul to favor intramolecular ligations. Wonder if that is what they are doing here?

--
Phillip

[lovebv]

Hello everyone，I constructed a mate pair library at the first time.The SOP is similar to 454 library pre protocol.But the size of the library I constructed was between 100bp-200bp.I wanted size of libraries to be 500bp.I cann't figure out why I got such results.Hope someone help me solve the problem.Thank you very much!

Sorry for the late response. Did you try increasing the Nick Translation time, from 11 mins? I tested out increasing nick translation time to 14mins and got an average library size of 350-400bp.

[Liting]

Hello

Yes, major size selection is during gel purification. There are AMPure clean up as well as column clean up to clean up after reactions. Good point, I will look into the % recovery at each step.

hello,Lovebv.I constructed a mate pair library at the first time.The SOP is similar to 454 library pre protocol.But the size of the library I constructed was between 100bp-200bp.I cann't figure out why I got such results.Hope you help me solve the problem.Thank you very much!

[Liting]

Hello everyone，I constructed a mate pair library at the first time.The SOP is similar to 454 library pre protocol.But the size of the library I constructed was between 100bp-200bp.I wanted size of libraries to be 500bp.I cann't figure out why I got such results.Hope someone help me solve the problem.Thank you very much!

[lovebv]

I have not constructed a mate pair library yet. Is there a size selection step? If so that may be a good place to start trouble shooting.

Yes, major size selection is during gel purification. There are AMPure clean up as well as column clean up to clean up after reactions. Good point, I will look into the % recovery at each step.

[asaleh]

I have not constructed a mate pair library yet. Is there a size selection step? If so that may be a good place to start trouble shooting.

[lovebv]

I only make the libraries and we send our libraries to SOLiD for sequencing. We're still in the testing phase, still deciding if the machine is worth the investment. We're a big sequencing center with other sequencing machines already... still debating if we 'really' need a SOLiD. My goal is to make larger insert MP libraries, 10KB and larger. We don't really need 3KB or smaller which is supported by the SOP.

The 5500 MP SOP is much different than SOliD 4. This is a ligation free process, I believe the library prep is the same idea, but the process has changed.

So far, I have to change the PCR cycle from 13 to 18 just to get enough library material. I'm not sure as to where I am losing material.

[asaleh]

we have a solid 4...Hope to get our 5500 in the next few mounts. My understanding is that library prep should not change much.

[VanessaS]

I'll be doing one library soon, I look forward to hearing how you have optimized the protocol. Have you run any of the libraries on the instrument yet?