Uppsala-Sweden/18 August 2009

Transforming ADH 2 Z.mobilis
The problems with transformaion of ADH 2 z have probably been due to insifficient overhang after the Pstl restriction site. To avoid this we now use the EcoRI and Spel restriction sites on the plasmid pSB1A3 and insert.

Digestion and Ligation was performed in two separate ways. The first on according to our standard procedure and the other with "on column" digestion. The "on column" digestion was performed by running a NucleoSpin PCR clean up without elution, this to bind the insert DNA to the column. Then the digestion was performed by pipetting 20µl of digestion mix with enzymes (Fermentas fast digest in amount recommended by protocol) on to the filter, this was then incubated and followed by a second and complete NS PCR clean up. --Flormane 12:24, 19 August 2009 (UTC)

Preparing Competent cells
Continued since yesterday

Three colonies from the Top-10 plate prepared yesterday was picked and inoculated into 2ml SOB and incubated on a shake table at room temperature over night. -Flormane 12:24, 19 August 2009 (UTC)

Taq grad PCR with pirA primer variations
Different primer variations were tested to find the non-functional or trouble causing primer. Template : Phusion PCR product of v2F + v2R


 * 1) pirAF + v2R : 1st@43,1°C | 2nd@44,7°C
 * 2) v2F + pirAR : 1st@46,6°C | 2nd@48,0°C
 * 3) pirAF + pirAR : 1st@55,2°C | 2nd@60,3°C
 * 4) v2F + v2R : 1st@43,1°C | 2nd@44,1°C

--Karl.brune 16:04, 19 August 2009 (UTC)

Phusion PCR on adh2(Y) and pirA (BB & grad)
I performed a Phusion PCR on adh2(Y) and pirA (BB & grad) For furhter information, have a look at the gel from 2009-08-19 (tomorrow)

--Karl.brune 16:04, 19 August 2009 (UTC)