From 2012.igem.org
Team:Technion/4_July_2012
Ilya
The concentration of the fusion PCR product (marked F1) is 25.5 ng/ul. I diluted it (marked F2) to a concentration of 5.1 ng/ul and used 2 ul for the next PCR reaction (elongation time 1 min, Ta=65) to add the prefix and suffix. When taking a photo of the gel of the PCR product we noticed there is a dominant band at ~300bp but we still got a band at ~850bp (as expected). Seems like this last PCR reaction was problematic. I still ran the rest of the PCR product for gel purification. When I was cutting the band at ~850bp out from the gel, I messed up. So all of today's work went down the drain. I will have to repeat this reaction. The product will be marked as Fus1.
After talking to Roee, we decided to repeat the last PCR reaction to add the prefix and suffix. Maybe use a higher Ta and GC buffer instead of HF buffer (which I used for the last reaction). Furthermore, Roee suggested we repeat the fusion reaction with a new AS primer, complementary to the end of mCherry and add a restriction site downstream to the gene. I figured out today that there is a tac promoter upstream to the riboswitch in ~1200bp product and there is an MCS including EcoRI upstream to that. That is why I chose to add PstI downstream to the gene. That way, we can clone it into pSB1C3 and test the riboswitch immediately. The product of this PCR reaction will be marked Fus2
I plan to do both PCR reactions once I finish the tests on 17/7. In the meantime it would be great if someone will transform BBa_J23119 (for testing the fusion product with prefix and suffix) and BBa_I0462 (which we will need later on) from the distribution kit, miniprep the plasmids and make glycerol stocks of these two biobricks. That way, once I do the PCR reactions, we can start the cloning steps.
To sum up our plan:
[ ]Design and order new AS primer to add PstI restriction site to mCherry.
[ ]Repeat fusion with S primer as before and new AS primer to produce Fus2 (expected ~1200bp).
[ ]Repeat PCR to add restriction sites, maybe with higher Ta, to produce Fus1 (expected ~850bp).
[ ]Clone Fus1 and Fus2 PCR products into pSB1C3 using EcoRI and PstI.
[ ]Clone Fus1 downstream to BBa_J23119 and BBa_R0062 using SpeI/XbaI and PstI.
[ ]Transform Fus2 into e. coli without lacI or use IPTG and test riboswitch.
[ ]Transform Fus1 into e. coli and test riboswitch.
Inbal
Asaf
Hila
Lior
Noa
Evgeni
Shahar
Rachel
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