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Sequencing Primers and Plasmids

DNA Sequencing Primers Reverse Forward Plasmid

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3 replies to this topic

#1 Epigeneticist

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Posted 08 January 2013 - 07:31 PM

I'm a little confused about the notation of the sequencing primers listed on the plasmid map for Invitrogen's pCR 2.1 and 4.0 used in TOPO TA cloning. Why is the forward primer 3' insertion site while the reverse primer 5' insertion. I thought it is opposite of that. Looking for some explanation. Wondering if I'm looking at the map wrong, or if I'm not understanding something. Link for map is below.

http://tools.invitro...cr4topo_map.pdf

For example:
Plasmid, M13 primer that is 5' to the insert site is called the reverse primer and the M13 primer that is 3' to the insert is called the forward primer.
PCR, forward primer has the same sequence as the 5' to 3' template and is 5' to the region being amplifying. While the reverse primer sequence is complementary to the 5' to 3' sequence and is 3' to the region being amplified. Seems opposite of sequencing primer notation.

I used the forward sequencing primer the first time and the sequence matched my reference but when I sequenced with the T3 priming site primer (5' to the insert) I got the complementary sequence to my reference.

Thanks

Edited by Epigeneticist, 08 January 2013 - 09:18 PM.


#2 Trof

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Posted 09 January 2013 - 09:32 AM

The map is OK.

The M13 primers are common for many (if not almost all current vectors) and have a historical identical notation 'M13 forward', 'M13 reverse'.
If those primers are actually in forward or reverse orientation on a plasmid map have no importance at all.
In TOPO cloning the insert is ligated randomly in both orientations (the more it doesn't really matter which primer you call forward and which reverse). So in the one clone you sequenced, insert was in "reverse" orientation regarding to the map.
That's all.

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#3 phage434

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Posted 09 January 2013 - 07:08 PM

Just a quick note: there are several versions of both the M13 forward and M13 reverse primers, which differ significantly in sequence. Use care.

#4 Trof

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Posted 11 January 2013 - 03:54 AM

@phage434: M13 primers are included with the kit, and the plasmid map specifies the sequence.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon






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