6 replies to this topic

[Ewan McGregor]

Hi!

I am needing help cloning a sequence into a vector.
I have this 1.5 Kb sequence to which I want to add 3 bases before the start codon.
I had a primer forward to amplify this sequence.
what I did to add these 3 bases was just adding 3 bases in the primer sequence like shown below:

Old primer was: 5' ctt a'gg ctt atg gcg aac ctt ggc tac - 3'
New primer:      5' ctt a'ag ctt (atc) atg gcg aac ctt ggc tac 3'

The difference between the two primers is the atc added just before atg.
After sequencing the plasmid extrated from postive colonies of top 10 cells transformed with pgem vector to which the pcr insert was ligated, I didn't have the sequence with the atc, but the sequence was as if I didn't change anything.
Now I don't have a clue of what happened, I wonder if there was a loop in the primer, as the rest could anneal but not the atc.
May be a contamination, but I don't think so. would it be better to remove the ctt just before the atg in the original primer and replace it with atc instead of addind the atc?
or in your experience what do you thing was wrong?

What would you do if it were you doing this??
please read and help.

Edited by Ewan McGregor, 23 March 2010 - 02:14 PM.

[Rsm]

Did you use a vector carrying your gene as template? If that vector has also ampicillin resistance, your bacteria will take up your original vector, and not the pGEM one.

Cheers,

Minna

[Ewan McGregor]

Minna, on Mar 23 2010, 07:08 PM, said:

Did you use a vector carrying your gene as template? If that vector has also ampicillin resistance, your bacteria will take up your original vector, and not the pGEM one.

Cheers,

Minna

When I select my colonies I pick the white one, but I had blue ones. doesn't it mean that it picked the pgem ones?

[tea-test]

Hi, I don't think it is a good idea to add new nucleotides in the middle of the primer. If you want to have a certain nucleotide sequence inserted by PCR you should add it at the 5' end. so start your new primer with ATC ATG GCG AAC... and add the appropriate bases at the 3' end to match the Tm.

[Ewan McGregor]

tea-test, on Mar 24 2010, 05:56 AM, said:

Hi, I don't think it is a good idea to add new nucleotides in the middle of the primer. If you want to have a certain nucleotide sequence inserted by PCR you should add it at the 5' end. so start your new primer with ATC ATG GCG AAC... and add the appropriate bases at the 3' end to match the Tm.

Well it was done that way because I needed the recognition site for HindIII

[Rsm]

Oh, I see... I haven't realized that you also inserted one "a" for HindIII, I thought it was about the "atc". Well, I think you can use your second primer directly, but you have to decrease the annealing temperature compared to the first primer. You've got 18 bases 3' overlapping with wildtype gene, maybe you should increase that to 20nt.
My initial question remains: did you use a vector as template, or cDNA library, or other? If you used a vector with Ampicillin resistance as template, almost all of your white colonies will be from that vector, and not the pGEM one.

Cheers,

Minna

[gyma]

dont use the old vector as pcr template and that will make things very easy. use cdna as the template and a high-fidelity polymerase to amplify the 1.5kb fragment, i am sure you will get the exact sequence you want and it wont generate any mutations for this length. so, it seems like you got contaminations by old vector. you said you dont think so. may i ask why?
how did you sequence the clones? are you sure you used the right primers for the sequencing?