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Old 07-20-2010, 11:16 AM   #1
454User
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Location: Mid-Atlantic, USA

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Default LMW band in paired end libraries?

Hello all,

To those of you that prepare Ti 3 and 8 kb PE libraries, have you ever experienced a prominent, low molecular weight band around 100-110 nt in your ssDNA product? Please see the attached images for examples.

I have seen this band without fail in many PE preps that span reagent lots and operator experience. While its abundance relative to the major library band varies, its presence does not. I am of the mind that this band represents:

1.) Library adaptor dimers (though these adaptors are not phosphorylated and I would not expect PNK to carry through the end polish cleanup)

or

2.) Amplification primer dimers that display this size due to their %GC

Any comments or insight would be greatly appreciated!

-J
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File Type: jpg 3kbPE1.JPG (32.6 KB, 27 views)
File Type: jpg 3kbPE2.JPG (36.7 KB, 17 views)
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Old 07-23-2010, 04:00 AM   #2
gilly
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Default

Hi.

Yes we do see them occasionally I believe the cause is amplification primer dimmers. If you take these libraries into emPCR then you run the risk of getting high enrichment with an increase in very short or even null beads.

This peak can be removed by performing a 4-cycle PCR, using identical conditions to those listed in the paired end manual (except with the cycle number reduced to 4 obviously). This step is to double-strand all of your products. This is then followed by a standard SPRI clean up (1.8x SPRI to sample ratio, as per Agencourt’s protocol).
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