IGEM:UNAM LCG/2009/Notebook/Hydrobium etli/2011/09/03
|iGEM Hydrobium etli|| Main project page|
Promoters PCR amplification
I made a PCR to amplify and extract the promoters: J23101, J23102, J23103, J23104, J23107, J23108, J23109, J23110, J23113, J23114, J23116.
It all went well, now I have all the Anderson Collection in a construction flanked by the standar preffix and suffix. The construction includes a RBS an RFP and a doble terminator at the end (aprox 1 kb in total).
The next step is to digest the constructions with EcoRI and PstI in order to ligate them with the plasmid pBBR1MCS5 backbone.
To make the PCR I used the Invitrogen's™ Platinum® Taq DNA polymerase, to define the reactives consentrations and PCR steps I checked out the manual online for the enzyme: PCR taq platinum manual.
I made 11 reactions (one for each promoter to amplify), the next table shows the reactives ammounts I used for a single reaction.
I ran a gel to check out the result of the PCR and the final concentration of the products.
1. J23101 2. J23102 3. J23103 4. J23104 5. J23107 6. J23108 7. J23109 8. J23110 9. J23113 10. J23114 11. J23116 12. Ladder //cuál ladder? The remaining lanes are not of interest for this analysis
By the lines we can say that all the parts were succesfully ammplified, they are all near 1kb which is the expected size and they are all near a quantity of 120ng (the dark lines un the ladder correpond to 60ng) except for J23104 (4th line) which appears to be 50ng aprox.