A question related to "reading" a vector

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bbs_r
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A question related to "reading" a vector

Post by bbs_r » Thu Apr 06, 2006 1:25 pm

Hi there. I just have 2 noob questions today:
1) for a vector map like this, should I look at the above sequence (red arrow 1) or the below sequence (red arrow 2)?

2) If I used the T7 primer for sequencing, does the sequence read in the direction like the blue arrows?
I mean the sequence will read as:
ATTATGCTGAGTGA.....


Thank you very much!
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Post by Trev » Thu Apr 06, 2006 3:13 pm

1. You should look at the ebove sequence, because below sequence is only complementary chain.
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Post by bbs_r » Thu Apr 06, 2006 6:31 pm

Thanks for your reply, but I just found something very strange, which is:
The pCRII T7 primer site sequence does not match anywhere with the official pCRII vector sequence. Help!

Official pCRII vector link:
http://www.invitrogen.com/search.cfm?searchTerm=pcrii&num=10&category=Vectors

pCRII vector Sequence:
http://www.invitrogen.com/content/sfs/vectors/pcrii_seq.txt

pCRII vector Map:
http://www.invitrogen.com/content/sfs/vectors/pcrii_map.pdf

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Post by canalon » Thu Apr 06, 2006 6:34 pm

1- Who cares? I mean as long as you know what side you are reading. But the top sequence should be read left to right, while the down one should be read right to left.

2- Well with this vector, you are supposed to use the M13 primers for sequencing. But if you insist on T7 promoter, your primer will TAA TAC GAC TC... and you results will be on the same strand so just before your insert you wil read TTC GGC TT(insert).
And if you can afford it it would be nice to sequence using both M13 primers. You will increase the possibility to have nice reads of your sequence. Yoou will be able to verify results, and if your insert is long it increase probabillity to have the complete sequence.
Patrick

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Post by Trev » Thu Apr 06, 2006 7:42 pm

Canalon wrote:... and if your insert is long it increase probabillity to have the complete sequence.

why? she (she? :) ) will design new pare primers and continue sequncing. it`s routine practice.
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Post by canalon » Thu Apr 06, 2006 10:41 pm

Trev wrote:
Canalon wrote:... and if your insert is long it increase probabillity to have the complete sequence.

why? she (she? :) ) will design new pare primers and continue sequncing. it`s routine practice.

Hum. Because if you can sequence in both direction without waiting to get first sequence, design new primers, order new primers, have them backordered, receive a mail telling you there is a QC problem and the new one will be delivered soon to finally run the second reaction and see that you reach the second end of the vector... it's easier to use the two M13 primers (usually found in all good sequencing departement, and delivered with the PCR II transformation kit) to get 2 sequences and have results earlier. Of course if your insert is very long you will have to do what you suggest, but when I know the 2 ends, I really don't see why not to take advantage of this situation. No?
Patrick

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Post by Trev » Fri Apr 07, 2006 9:47 pm

ok :)
.
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