Team:Technion/3 September 2012




- Miniprep of the low copy number pCP plasmid.
- PCR of Fus2 and Fus2del from pSB3C5.


- Mini-prep for the 3 starters I put yesterday. The concentrations are: 115, 197 and 150 ng/ul! :] (at least I know how to mini prep like a pro!)
- 4 starters of BBa_B0015 ( the insert is double terminators, the size is 2200bp roughly).
- Cleaning from gel the T7* RNAP (the one with pSB1A2 primers), the concentrations were l-o-w: about 7.5ng/ul after calculating it from just looking at the band's intensity (cause the NanoDrop wasn't accurate).
- clean the pSB1C3 plasmid after digestion with XbaI and PstI by cleaning kit.


I cleaned the PCR products of the polymerase genes from the gel.


PCR reaction for fragments 1, 1C, 6, 6C using T = 55, 58, 60.
Cross your fingers…

Restriction with PacI and SpeI for fragments 2C, 3C, 4C, 5C, 7C, 8C and the confirmed MCS in the pSB1C3+MCS.




- No growth of starters again!!! Probably I'm doing some stupid mistake during their preparation, Orna will watch over me today during starters' preparation.


-Clean the N$ & T3 RNAP after digestion by cleaning kit.
-Those inserts (N4 & T3 RNAP) are ready for cloning into pPROLAR plasmid.


-Clean the PCR product of K1F RNAP (the one with pPROLAR primers)from the gel. The concentration was: 17 ng/ul.
-Digest of K1F RNAP with HindIII and NHeI for creating sticky ends.
-Clean the K1F RNAP after digestio by cleaning kit. The final concentration: 25 ng/ul
-This insert (K1F RNAP) is ready for cloning into pPROLAR plasmid.