Login

Join for Free!
118235 members


Help: Confused with primers in a PCR of a vector?

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

Help: Confused with primers in a PCR of a vector?

Postby keetner » Sat Mar 15, 2014 3:21 am

Hi guys, I am an undergrad being introduced to a lot of different lab techniques, so I'm not entirely familiar with what is going on.
We recently ligated a gene into a pBS vector and popped it into some E. colicells. We looked for positive clones via blue/white screening and isolated/purified the respective plasmid. Then, we carried out a PCR reaction of the pBS-gene (our clone). This PCR reaction is something I do not really understand.

We used the pBS-gene as our template. We carried out four reactions, which are as followed:

1) T7 (forward primer); T3 (reverse primer)
2) T7 (forward primer); coding reverse (reverse primer)
3) T7 (forward primer); coding forward (reverse primer)
4) T3 (forward primer); coding reverse (reverse primer)

Here is an image of the vector we used: http://www.bio.davidson.edu/courses/mol ... ipt%2B.gif. On the right hand side, it says, "MCS" (where our gene of interest is located), of which is "flanked" by the T3 and T7 promoters. The thing that throws me off is just where these primers will bind to, and what the subsequent product will be. The one that really throws me off is number 2, because it seems like we might not get a reaction? Are number 1 and 2 just positive and negative controls?

Based on the few PCR reactions I tried drawing out myself (sorry, don't have an image), from my understanding, the amplified products I should get are:

1) T3 - coding region - T7
2) ??? It looks like it WON'T be a proper PCR reaction? Will we just get one massive chunk of DNA?
3) coding region - T7
4) T3 - coding region

I think the point of this entire process was to just determine the directionality of our insert. Because later in the lab, we did a whole bunch of sequence alignments with our gene (of both strands) and BLASTed it. But...I am just not really sure if I've done the PCR reactions correctly.

Any help would be greatly appreciated. Thanks!
keetner
Garter
Garter
 
Posts: 14
Joined: Sun Sep 30, 2012 4:44 am

Postby poobear » Fri Apr 11, 2014 9:58 pm

1) positive control (T7-gene-T3).

2) if your gene is inserted into the vector so the beginning of the gene is right next to T7: T7-gene.
If your gene is inserted into the vector so that the beginning of the gene is next to T3: Nothing since both primers bind in the same direction.

3) if your gene is inserted into the vector so the beginning of the gene is right next to T7: Nothing since both primers bind in the same direction.
If your gene is inserted into the vector so that the beginning of the gene is next to T3: T7-gene.

4) if your gene is inserted into the vector so the beginning of the gene is right next to T7: Nothing since both primers bind in the same direction.
If your gene is inserted into the vector so that the beginning of the gene is next to T3: gene-T3.
poobear
Garter
Garter
 
Posts: 23
Joined: Thu Nov 06, 2008 5:33 pm

Postby JackBean » Sun May 04, 2014 12:00 pm

It's basically designed so that you see both length of your insert and it's direction. Did you use blunt-end cloning? Because otherwise one doesn't need to be so careful about the directions.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5678
Joined: Mon Sep 14, 2009 7:12 pm



Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 3 guests