IGEM:Harvard/2007/Laboratory Notebooks/Quorum Sensing/Week 5
Colony PCR of R-P constructs
Stephanie Colony PCR'ed the following:
- R0052<P0140 colony #1 (worked)
- R0052<P0140 colony #2
- R0051<P0140 colony #1 (worked)
- R0051<P0140 colony #2 (worked)
- R0051<P0340 colony #1 (faint)
- R0051<P0340 colony #2
- R0011<P0340 colony #1
- R0011<P0340 colony #2 (worked)
- R0052<P0340 colony #1 (worked)
- R0052<P0340 colony #2
- R0011<P0140 colony #1
Plate Reader (Light Scattering)
Stephanie set up another plate reader for fluorescence. One of the things she is testing is choosing ridiculous wavelengths to see if we can get light scattering data that correlates to cell growth.
Miniprep of the J04450 and JT constructs
In preparation for building the constitutive RFP + JT construct, George miniprepped the following parts which were grown overnight:
Digesting the J04450 and J-T
Sequencing the Promoters: R0011, R0051, R0052
Dephosphorylating the J04450 and J-T
George dephosphorylated the J04450 and the J-T with Antarctic Phosphotase and then heat inactivated the AP. Silly George, AP is for vectors not inserts. Redo it tomorrow.
Liquid Cultures of R-P constructs and J-T
George grew liquid cultures of the following:
- R0011 <P0340 colony #2
- R0051 <P0140 colony #1
- R0051 <P0340 colony #1
- R0052 <P0140 colony #2 Lower Right
- R0052 <P0340 colony #1 Upper Right
- R0052 <P0340 colony #1 Lower Right
- J23039 <T9002
The Upper/Lower Right is due to the fact that the colony PCR plate had duplicates of those colonies and it was unclear which colony was correct (at least, the PCR and gel was correct). So, George took both samples and labeled one as coming from the upper right of the plate (when viewed with the handwriting going the correct way) versus lower right.
Miniprepping the R-P and JT
Digesting the R-P and JT
Sequencing the R-P constructs
George sent off sequences for the R-P constructs
Dephosphorylating the R-P constructs
George dephosphorylated the R-P constructs with Antarctic Phosphotase and then he heat inactivated the AP.
Clonewell and Vacufuge of R-P constructs and JT
George clonewelled and vacufuged the R-P constructs. Unfortunately, he forgot to include JT and had to re-use the same gel.
Ligation and Transformation tetR-JT and RFP-JT constructs
George Roche ligated the following:
and then transformed them into BL21.
Colony PCR of tetR-JT and RFP-JT
George colony PCR'ed the tetR-JT and RFP-JT constructs.
Liquid Cultures of R-P and JT parts
George grew liquid cultures of all the working R-P parts (according to the colony PCR). He discovered that the R52's were incorrect (sequences + wrong selection plate). The parts he grew up were:
- R0051 <P0140 colony #1 in Top10
- R0051 <P0340 colony #1 in Top10
- R0011 <P0340 colony #2 in Top10
- J23039 <T9002 in BL21
Miniprep of R-P and JT
Stephanie miniprepped the parts that were grown in liquid culture.
Digestion of R-P and JT
Stephanie digested the parts that were miniprepped.
Dephosphorylation of R-P and JT
George dephosphorylated the JT with Antarctic Phosphotase.
Clonewell and Vacufuge of R-P and JT
George clonewelled and vacufuged the R-P and JT
Colony PCR of RP-JT parts
George colony PCR'ed the RP-JT parts for the third time. Unfortunately, the results were horrible.