From 2012.igem.org
Team:Technion/18_June_2012
Ilya
We didn't get any product again for the PCR reaction to add the restriction sites. After talking to Roee, we decided to troubleshoot the process. We set up three reactions:
1) With the two primers we used in the fusion reaction. This is a control to see if we can amplify the 1247bp fragment of RS+mCherry.
2) The sense primer for the RS+prefix for restriction sites and the antisense primer we used in the fusion PCR (expected 818bp fragment).
3) The sense primer we used in the fusion PCR and the antisense primer for mCh+suffix for restriction sites (expected 1276bp fragment)
All reactions ran with Ta=58 degrees and elongation time 24 secs. 9.75 ng of the purified ~1200bp fragment from the fusion PCR gel was used as the template in a 50 ul reaction.
I ran the PCR products on a gel and got the following results. In reactions 1 and 2 there was a band a little bigger than 500bp and a primer cloud. In reaction 2 there was only a strong primer cloud.
Reaction 2 is different from 1 and 3 in the sense primer. It seems something is being amplified as long as the sense primer is the one used for the fusion (homological to the puc19 part upstream of the RS). This fragment which is a little bigger than ~500bp was also dominant in the fusion PCR reaction from which we gel purified our ~1200bp fragment. This makes me believe that there are secondary structures in the fused sequence which make the polymerase drop at ~500bp.
I have a few ideas for what to do to continue troubleshooting:
1) Run the products on a gel again along with the amplified mCherry product, puc19+RS fragment and the ~1200bp purified band from the fusion PCR reaction.
2) Repeat the PCR reaction with primers from the fusion PCR reaction but this time use GC buffer.
3) Repeat the PCR reaction with primers from the fusion PCR reaction but this time use GC buffer and add DMSO.
4) Repeat the fusion PCR reaction with GC buffer and maybe DMSO. Split the reaction into two parts. First do a two step amplification without primers to fuse the two fragments. Next, add the primers and more DNTPs and do regular PCR cycles to amplify the fused fragment.
In the meantime we should try and start moving on with any cloning steps we can do in parallel to troubleshooting of the fusion PCR. We need the RS+T3 RNAP from Omri as soon as possible in order to clone it upstream to the RBS+LuxR+terminator biobrick in pSB1A2 (we will clone it into pSB1C3 with the other parts later on).
Inbal
Asaf
Hila
Lior
Noa
Evgeni
Shahar
Rachel
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