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Old 02-01-2012, 02:54 PM   #1
melcar
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Default problems with M13 universal tailed fusion PCR for 454 pyrosequencing

I am having great difficulty getting the 2nd round PCRs (where I add the 454 fusion tailed primers to my template specific primers using an M13 primer tail) working effectively. I generally get a very low amount of PCR product that is often a smear. I have been gel purifying the 1st round add trying a variety of PCR protocols and profiles (adding 150ng 1st round template and PCRing for 4 cycles and also just adding 1ul and PCRing for 15 cycles), but nothing seems to work. Does anyone have a protocol that works for this step?
I am also concerned that the overhanging "A" added by the taq polymerase may be a issue for annealing of fusion tailed primers. Has anybody found the type polymerase has an effect? I have tried Bio-act short mix and Roche's FastStart.

Any advice would be most welcome!
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Old 02-13-2012, 05:39 AM   #2
neophyte
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Maybe a "suppressive PCR" effect ?
see Evrogen...
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Old 05-16-2012, 07:55 AM   #3
ParkerKB
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I am just about to embark on this so don't have much to offer...have you seen Bybee et al. 2011 and Hajibabaei et al. 2011? Bybee uses a different tail and Hajibabaei doesn't specify, but I am assuming it is also M13 as I know they use M13 tails for standard sanger sequencing. Daigle et al. 2011 also has some details. I also recently came across a blog of someone who is troubleshooting this, I haven't read it thoroughly but it may have some ideas.

http://enggen-nau.blogspot.ca/2012/0...es-off-to.html
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File Type: pdf Hajibabaei et al. 2011.pdf (1.26 MB, 35 views)
File Type: pdf Daigle et al. 2011.pdf (317.9 KB, 29 views)
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Old 06-04-2012, 06:55 PM   #4
melcar
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Thanks for the tips. I ended up having alot of trouble using an m13 tail with my COI primers. I ended up developing my own tail, which was G-C rich. This meant I could use a high Tm and avoid problems with secondary structure in the 2nd round PCR's. Although I haven't got the data from the run yet to see how well it has worked

It is hard to find detailed information on the success of different universal tails
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Old 05-07-2013, 05:16 PM   #5
lotijeo
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Default universal tail

Hi Melcar,
Did you have any success using customized universal tails? I wanted to try the M13 sequences but maybe you had a better luck with your customized GC rich tail?
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Old 05-07-2013, 11:26 PM   #6
melcar
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The GC rich Tail work really well, I got heaps of product in the secondary PCR and it sequenced well using 454. I would definitely use these types of primers over M13. I have submitted the work for publication, so finger crossed it will be out soon
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Old 08-11-2013, 06:11 AM   #7
alezhe
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melcar

we would like to know details of your success. could you share with the public the sequences of universal tails you have developed.
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Old 08-11-2013, 02:42 PM   #8
melcar
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Default Universal tail

The work in now published. See below

Title: Environmental monitoring using next generation sequencing: rapid identification of macroinvertebrate bioindicator species

Authors: Carew E Melissa, Pettigrove J Vincent, Metzeling Leon, Hoffmann A Ary,

DOI: 10.1186/10.1186/1742-9994-10-45

URL: http://www.frontiersinzoology.com/content/10/1/45

You will find the sequences for the universal tails in the methods

Cheers
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Old 08-11-2013, 02:44 PM   #9
alezhe
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Thank you a lot!
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