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Old 02-20-2019, 09:42 AM   #1
Adem80
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Default Why is the ME-rev oligo on Tn5 phosphorylated?

Hello all,

Does anyone know why the oligo named ME-rev, annealed to ME-A and ME-B before being loaded on the Tn5, is phosphorylated at the 5' end ([phos]CTGTCTCTTATACACATCT)?

Thanks a lot.
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Old 02-20-2019, 08:45 PM   #2
nucacidhunter
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Nextera (modified Tn5 transposase) inserts ME sequences by cut and paste mechanism and leaves a 9 base gap at the 3' end of insertion site. The gap is filled during 3 min incubation at 72C at PCR step. If there is no 5' phosphate there will be a nick and PCR will fail.
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Old 02-20-2019, 09:25 PM   #3
Adem80
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Thanks nucacidhunter.

But the Tm of this short fragment is 45C. At 72C it is probably detached from the transferred strand.
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Old 02-21-2019, 01:18 AM   #4
nucacidhunter
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The calculated Tm is correct. Empirically it is difficult to dissociate that oligo. For an application I tried to inhibit gap filing by both heat and a chemical method. PCR yield was decreased by 65% but was not inhibited completely.
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Old 02-21-2019, 03:10 AM   #5
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Thank you very much.
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