problem with the ligation of adaptator- Rapid library

doc_lille · 07-27-2010, 07:25 AM

Dear All,
In the aim of a prokaryotic RNA sequencing on Roche 454; I used the kit Rapid cDNA Library (GS FLX Titanium).
To recover the small RNA, I modified slightly the protocol at the level of purification (calibration of beads Ampur) between the enzymatic steps (RT, Fragment repair, ..)
Unfortunately, it seems that the step of the ligation of double-stranded adapters doesn't work very well because the controls checked using Bioanalyser of Agilent (high sensitivity) show the presence of my product, but no matches were exploitable using the TBS fluorometer and at the stage of titration.

Has anyone already meet this kind of problem on the kit Rapid library?
thanks for your advices.
cheers.

pmiguel · 07-27-2010, 09:16 AM

Can you post your Bioanalyzer trace?

doc_lille · 07-28-2010, 12:21 AM

To be a little more precise, my starting material is composed exclusively of messenger RNA.
In order to detect if I effectively linked the adapters on the cDNA double stranded, I made a conventional PCR (35 cycles) with primers A / B (sequences ligated adapters) but my PCR still always negative even after an developpement concerning a gradient of the concentration in MgCl2 and gradient of temperatures.
thank you for hypothetical explanations

pmiguel · 07-28-2010, 05:41 AM

Did you do a positive control to make sure the primers you are using will work?

If so, then it is likely that your adaptors failed to ligate to the double stranded cDNA. Possible explanations for this would include:

(1) Contaminants from clean-up steps/washes (eg, chaotropes or alcohols) prevented one or more of the blunting, a-tailing or ligation reactions.
(2) First or second strand cDNA synthesis failed.
(3) exonucleases destroyed your adaptors during the ligation step.

The above is little more than my writing "either you QC assay has failed, or, indeed, you failed to produce amplicons".

--
Phillip

doc_lille · 07-28-2010, 08:04 AM

Dear phillip,
thank you for your quick and accurate response.
In fact I tested the primers A and B on a previous library prepared from a kit GS FLX Titanium (not quick Libary preparation) and saw a good smear on the agarose gel after the PCR.
In terms of hypothesis No. 2, I do not think that it was a trouble with the RT or the double strand synthesis because the Bioanalyser (DNA Chip) posts a explanable profile, it is therefore clearly an dsDNA. Moreover, With the same RNA, i tested another kit from Ambion which included a RT and double strand synthesis, then after purification with Ampure, i came back into the Roche kit at the Fragment repair step until the end.
After all, i observed the same profil using the Bioanalyser and my PCR control A and B , still not working.
I keep in memory the prospective problem with the contaminant about the wash step, i will test this hypothesis using the MinElute kit (Qiagen ).
Keep in touch.
thanks

pmiguel · 07-28-2010, 09:14 AM

The most common issue I see with MinElute is ethanol contamination. This is easy to fix using a speed vap. If you nose is sensitive to ethanol, you can smell it.

Second most common, I think, are Acetate salts, I think. You can actually see these, if they are really high, in the UV spectrum as a peak in the 230 nm region.

--
Phillip

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07-27-2010, 07:25 AM	#1
doc_lille Junior Member Location: france Join Date: Mar 2009 Posts: 5	problem with the ligation of adaptator- Rapid library Dear All, In the aim of a prokaryotic RNA sequencing on Roche 454; I used the kit Rapid cDNA Library (GS FLX Titanium). To recover the small RNA, I modified slightly the protocol at the level of purification (calibration of beads Ampur) between the enzymatic steps (RT, Fragment repair, ..) Unfortunately, it seems that the step of the ligation of double-stranded adapters doesn't work very well because the controls checked using Bioanalyser of Agilent (high sensitivity) show the presence of my product, but no matches were exploitable using the TBS fluorometer and at the stage of titration. Has anyone already meet this kind of problem on the kit Rapid library? thanks for your advices. cheers.

07-27-2010, 09:16 AM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Can you post your Bioanalyzer trace?

07-28-2010, 05:41 AM	#4
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Did you do a positive control to make sure the primers you are using will work? If so, then it is likely that your adaptors failed to ligate to the double stranded cDNA. Possible explanations for this would include: (1) Contaminants from clean-up steps/washes (eg, chaotropes or alcohols) prevented one or more of the blunting, a-tailing or ligation reactions. (2) First or second strand cDNA synthesis failed. (3) exonucleases destroyed your adaptors during the ligation step. The above is little more than my writing "either you QC assay has failed, or, indeed, you failed to produce amplicons". -- Phillip

07-28-2010, 08:04 AM	#5
doc_lille Junior Member Location: france Join Date: Mar 2009 Posts: 5	Dear phillip, thank you for your quick and accurate response. In fact I tested the primers A and B on a previous library prepared from a kit GS FLX Titanium (not quick Libary preparation) and saw a good smear on the agarose gel after the PCR. In terms of hypothesis No. 2, I do not think that it was a trouble with the RT or the double strand synthesis because the Bioanalyser (DNA Chip) posts a explanable profile, it is therefore clearly an dsDNA. Moreover, With the same RNA, i tested another kit from Ambion which included a RT and double strand synthesis, then after purification with Ampure, i came back into the Roche kit at the Fragment repair step until the end. After all, i observed the same profil using the Bioanalyser and my PCR control A and B , still not working. I keep in memory the prospective problem with the contaminant about the wash step, i will test this hypothesis using the MinElute kit (Qiagen ). Keep in touch. thanks

07-28-2010, 09:14 AM	#6
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	The most common issue I see with MinElute is ethanol contamination. This is easy to fix using a speed vap. If you nose is sensitive to ethanol, you can smell it. Second most common, I think, are Acetate salts, I think. You can actually see these, if they are really high, in the UV spectrum as a peak in the 230 nm region. -- Phillip