Big insert to put back into the vector - (Feb/17/2009 )

I forgot to ask smthing:)

If I have about 33-34 bp which do not match anything, how many should I add from my seguence so it would work fine ? I had about 15bp but the Tm is very low and does not match the forward (20compared to 58), how long can the primer be, as you can image with only 15bp matching, if I put the whole Sma1 seguence it is 51bp:), and a couple of overganging...


Thank you,


Ana

XmaI needs at least 3bp flanking each end of the restriction site before the enzyme will cleave the restriction site efficiently. I would use at least 5bp to be on the safe side.

the primer can be as long as 100bp, the maximum that most companies will sell you. The tm of both primers must be as close as possible. As you have noted 15bp is too short. Make it longer, increase the length of the sequence until the tm of both forward and reverse primers are the same. I would aim for a tm of 58 Celsius.

Could you rephrase "If I have about 33-34 bp which do not match anything, how many should I add from my seguence so it would work fine ??". I am not certain what you mean. (Below is what I think you mean)

I assume that this 33-34bp block must be included and is inert as far as PCR goes. AKA this block doesn't bind to your template. The length of the sequence that does bind to the gene template is dependent on the tm rather than the length. I would aim for tm of 58 Celsius for all my primers. Thus a the section of the primer that bind to an AT rich template would be longer than a primer that bind to a GC rich template.

Yes,

That was what I meant, 30bp which I must put in but do not match my template.

But what about blunt ends? can I used them without adding any overhanging pb? so I wouldn't need a clean up afterwards


Thank you in advance

ups on Feb 17 2009, 02:51 PM said:

Yes. If you used blunt end cloning. You don't need to add any overhanging bp. However blunt end cloning is more difficult that sticky-end cloning. So whenever possible I would try to avoid blunt end cloning.

Your PCR amplified gene already has a EcoRI site on the 5' end. So you have to clean up the product regardless. Thus one might as well have a sticky end XmaI on the 3' end

perneseblue on Feb 17 2009, 11:25 PM said:

Sorry for all these quenstions, I have another Q: would it be more helpful if I would add after the 33bp non matching seguence, some bp that would match my template just after the gene of interest stops?

Sorry for the questions:(

Sorry... I made a mistake when referring to the 5' restriction site. I meant EcoRI... rather than BamHI which I said.

perneseblue on Feb 18 2009, 08:56 PM said:

Thank you very much for the schematic representation, I must say it was very suggestive:)

I am now trying to check for primer dimers, secondary structure dimers, and anything else that I should consider... I have tried to use Vector NTI, but it seems to me it is not that explicit when it comes to these details. Do you have any suggestions regarding the software that I can use to check for these details?

What else should I look for besides the primer dimer, sec str dimer ?

Sorry for the questions

Thank you again for the representation, very good I must say:)

Ana

I used northwestern calculator.

I check for tm, and try to minimise hairpin loop structure, and primer self annealing. A high tm (58 C to 60 C) is used to cover some of the deficiencies of the primer. the high tm causes these structures to melt.