I am working on RNAi of nematode genes for which I am adopting gateway cloning strategies. In this strategy to clone the gene in the entry vector (I am using pDONR221 from invitrogen) some adapter sequence (att B1 & B2) has to be added in our gene of interest. The only way to add those sequences in the upstream of forward (attB1) and reverse primer (attB2) before going for PCR. The problem I faced while designing the primer was invitrogen provided the sequence of attB1 is GGGGACAAGTTTGTACAAAAAAGCAGGCT. But when I checked in oligocalc software (http://www.basic.northwestern.edu/biotools/oligocalc.html) this one showed there is a potential of hairpin formation and self annealing site. so I am worried PCR may not work. Also primer length is going to be too high 29+20 bp (attB+gene specific) leading to high annealing temperature. Does anyone have any suggestions?
Hi,
I have been using gateway cloning quite a lot and I never ever had problem in PCR amplification with primers having these recombination sequences. I even have used successfully, longer primers than you. But if still you are not comfortable you can devide your priming sequence into to two overlaping primers. Also check carefully that you are not going to add any stop codon due to these sequences, if you are going to express you gene in bacteria.
I suggest doing a temperature gradient PCR from 55ªC-65ªC and also suggest you add 1% DMSO final volume to avoid the formation of secondary structures
Hello,
I have designed primer of 58-60 bp for BP reaction. using primestar GXL (from Takara ) to amplify my genes of interest, set 55 degree as annealing temperature for 30s. It worked quite well.
But I have another problems, have few genes of 5K-9K in length, performed BP reactiom many times but only get less than 4 clones those are negative.
Do you have any suggestions?
I am using this tecnique since one year. There is no such problem as you described earlier. The attB1 and attB2 are the 31 and 30 nucleotide sequences which are attached to your primers when you design your primer using VectorNTI. The generated primers will have a size of 30 or 31 + your actual primer i. e around 50 nucleotide. Upto best of my knowledge attB1 and attB2 never reported for making hairpin structure. For PCR amplification using such long primer (Gateway primers) , generally use tm of actual primer excluding attB1/B2. if in such case tm is below 52 or gene is not being amplified or primer dimer is formed then you can try long PCR stating from 65 or 68 to 55 ot 53.Most of the time it works well. Ofcourse for such type of clining u should use phusion to avoid any mutation.
@Wei Chen , You can try electroporation. in this you get large number of colonies as compare to conventional method and that will increase the chance of getting positive clones.
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