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I've just started constructing 5500 Mate Paired libraries, the first few worked well but lately I have been unable to generate any library even though the trial PCR shows a band at 300bp. Has anyone else had this problem or any ideas? Thank you.
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increase the number of cycles for fullscale amplification. For some reason the trial PCR seems to work better. I almost always have to add a few more cycles to the full scale PCR. Maybe something with the beads inhibits PCR? -
Yes, I was told by a ABI FAS that the beads are inhibitory. One work around might be to denature the steptavidin and recover the library molecules in the absence of the beads. See:Originally posted by SeqAA View Post
Electrophoresis 2005, 26, 501–510, Anders Holmberg, Anna Blomstergren, Olof Nord, Morten Lukacs, Joakim Lundeberg, Mathias Uhlén, The biotin-streptavidin interaction can be reversiblybroken using water at elevated temperatures.
for details.
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PhillipComment
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Excellent. Thank you for feedback. I will give it a go.Comment
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Thank you!!! I was having the same issue, could never figure out what I was doing wrong as I feel the trial PCR was giving me false hope.Comment
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Final PCR Troubleshooting
Hi clissold,
How many cycles did you do your final amplification for? Typically you don't have to do that many cycles to get slighty more than a faint band with your remaining 26 uL of beads to do a lot of sequencing. I haven't had any problems with final amplifications as long as the trial PCR went well (easily identifiable bands for at least the 14 cycle trial PCR with low input, and a faint or strong band for the 10 cycle trial PCR depending on DNA input quantities). Did you see decent bands both at 10 and 14 cycles of trial PCR?
As long as you didn't forget the 20 minute/72 Celsius nick translation step for the final amplification (a new change in the 5500 protocol), and you used the same amplification mix and primers as you did for your successful trial PCR reactions, the results for the final amplification shouldn't be any different. However, the bead mass (26 uL of beads versus 4 uL of beads) for the final amplification is much greater for the final amp versus the trial PCR, so make sure you pulse vortex at full speed right before the final PCR, and do not pulse centrifuge to the point of bead pelleting. You can examine the PCR tube bottom before you put it into the thermocycler for the final amplification to see if any pelleting of beads occured during pulse centrifugation.Comment
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Just out of curiosity, does anyone know why the 20 min/72c nick translation is necessary?Comment
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Yes, that is strange.
Either the P1-T and P2-T adapters are not 5' phosphorylated which would be bizarre (the "T" overhands should prevent their ligating), or the insert is not 5' phosphorylated prior to adapter ligation?
The insert results from and earlier nick translation of circularized DNA followed by treatment with BstT7 nuclease and S1 nuclease. So the 5' end of the insert is generated by S1. S1 should remove 5' mononucleotides in single stranded regions, so I would expect the 5' phosphate to remain attached to insert.
I guess the 5' insert phosphate might be removed during dA tailing somehow?
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PhillipComment
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The P1-T and P2-T adapters are indeed not 5' phosphorylated that is why a nick-translation step is still necessary.Comment
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