Team:TUDelft/14 August 2009

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14 August 2009

Tim Weenink

First did ligation of !E and !F with the parts that were not excised from gel.

Component for !E volume (uL)
*S PCR (upstream) 2
 !C PCR (downstream) 2
pSB1AK3 from !A assembly 2
T4 ligase buffer 2
T4 ligase 1
H2O 11
Component for !F volume (uL)
 !B PCR (upstream) 2
 !D PCR (downstream) 2
pSB1AK3 from !A assembly 2
T4 ligase buffer 2
T4 ligase 1
H2O 11


Then I did electroporation of the !E and !F gel extracted assemblies.

After that I did chemical transformation of !E and !F assemblies (both gel extracted and direct PCR restricted fragments)

Calin

oriTR_KO +L-ara +PCR and trbK_KO +L-ara +PCR backup plates made on 0.5x KAN.

Did miniprep on CB and CC culture tubes.

Plate 2, 4, 6, and 8 are overgrown. Cells survived electroporation. No colonies on knockout plates or control plates.


Part DNA concentration 260/280 260/230
CB 57.7 2.17 2.30
CC 121.1 2.01 2.33

Ran 1.5% gel with CC and CB.

Gel140809.png

Well Part Expected Plasmid Size Status
1 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] 2953
2 CB 1162  ? too light
3 CC 311  ? SpeI not cutting

Digested CB with X + P (11uL DNA). Digested CC with E + S (4.5uL DNA).

Daniel

No growth although gel seems good. Let´s think about it during the weekend