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problem with my clone


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#1 jason0429

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Posted 12 November 2009 - 07:49 PM

Hi everyone, I have been doing this cloning for 3 months but I am not sure whether I've gotten the clone. Please help me.

I managed to ligate my insert into my vector, pET15b. I did PCR to screen for my clone using two sets of primers. One is my insert primers and another is my pET15b T7 primers. The PCR results showed me that with my insert primers, the PCR works showing me a 1000bp band which is correct. However, there are not results for my pET15b T7 primers. What is the reason??
Then i decided to do another PCR using different combination of the primers.

Primers Combinations:
1. T7 promoter & Insert Reverse
2. T7 terminator & Insert Forward
3. T7 promoter & T7 terminator
4. Insert Forward & Insert Reverse

The PCR results showed the first (1.) and forth(4.) primer combination works, but the second(2.) and third(3.) does not work. It seems like those combinations with T7 terminator primers is not working. But my positive control for T7 promoter & T7 terminator works perfectly well. what is the reason???

Then i tried to look at the sequences. It showed that both T7 promoter and terminator have 65% similarity with my insert, which this might cause the primer to bind to the similar region, but why does the first primer combination(1.) works only, but the second (2.) primer combination does not???

I have sequenced my clone using T7 promoter and terminator primers. With the T7 promoter primer the sequencing works perfectly well and there were no mutations with my insert. However, the T7 terminator primer did not show any sequencing results, no results were obtained, no signals.

What is the reason?? Why is the T7 terminator is not working???? Please help me...

Thanks....

#2 pesji

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Posted 13 November 2009 - 05:51 AM

Hi everyone, I have been doing this cloning for 3 months but I am not sure whether I've gotten the clone. Please help me.

I managed to ligate my insert into my vector, pET15b. I did PCR to screen for my clone using two sets of primers. One is my insert primers and another is my pET15b T7 primers. The PCR results showed me that with my insert primers, the PCR works showing me a 1000bp band which is correct. However, there are not results for my pET15b T7 primers. What is the reason??
Then i decided to do another PCR using different combination of the primers.

Primers Combinations:
1. T7 promoter & Insert Reverse
2. T7 terminator & Insert Forward
3. T7 promoter & T7 terminator
4. Insert Forward & Insert Reverse

The PCR results showed the first (1.) and forth(4.) primer combination works, but the second(2.) and third(3.) does not work. It seems like those combinations with T7 terminator primers is not working. But my positive control for T7 promoter & T7 terminator works perfectly well. what is the reason???

Then i tried to look at the sequences. It showed that both T7 promoter and terminator have 65% similarity with my insert, which this might cause the primer to bind to the similar region, but why does the first primer combination(1.) works only, but the second (2.) primer combination does not???

I have sequenced my clone using T7 promoter and terminator primers. With the T7 promoter primer the sequencing works perfectly well and there were no mutations with my insert. However, the T7 terminator primer did not show any sequencing results, no results were obtained, no signals.

What is the reason?? Why is the T7 terminator is not working???? Please help me...

Thanks....


I remember that some T7 primers are different from plamid to plasmids be sure to verify that you use the correct primers: Another thing you can try is to use another primer located outside of this T7 region just to check:

Due to this T7 primers problems I usually use only the M13 universal primers for PCR

Hope it helps

#3 jason0429

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Posted 14 November 2009 - 11:21 PM

Hi, thanks for replying.

Does pET15b contain M13 universal primer??? Where is the M13 in the pET15b vector??

So do you think i should continue and try to express my protein???

#4 pesji

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Posted 15 November 2009 - 12:55 AM

Hi, thanks for replying.

Does pET15b contain M13 universal primer??? Where is the M13 in the pET15b vector??

So do you think i should continue and try to express my protein???


I check the vector map and effectively there's no M13 sequence but why not trying a small scale purification of the clones ? Set up a small culture and do a induced versus non-induced culture and lyse the pellet by SDS page buffer and check on a SDS PAGE you'll see immediately




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