Team:TUDelft/11 August 2009

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11 August 2009

Tim Weenink

imaged the gel i ran yesterday:

Tim110809!B!C!S.png

This is a really ugly gel, because the comb for creating the wells was too long. The bottom of the wells was too thin. Upon removing the comb, some of the bottoms were damaged, making them leak the loaded samples.

The contents of the lanes is as follows:

Calin

Growth in all tubes, miniprep was done.

Growth on all plates.

Checked concentrations from yesterday:

Part DNA concentration
trbK 1.9
pSB1A3 5.2
pSB4A5 5.2
pSB1C3 3.5
GFP-gen 1.9
oriT-R 4.0

Make R751 wild cells electrocompetent using Knight's procedure.

Did E + P digest on trbK. Did ligation for assembly CB (GFP-gen + oriT-R on pSB1C3) and CE (trbK on pSB1A3).

Made 3 new AMP plates for pKD3, pKD4, and pKD46.

Transformed electrocompetent cells with CB (CAM, timeconstant 4.6) and CE (AMP, timeconstant 4.5) assemblies. Both plates and culture tubes were made. Transformed R751 electrocompetent cells with pKD46, timeconstant 4.2 and left plate in 30C.

Ran a trbK digest with X + P, this was loaded into the last lane in todays 8 well 1% gel and extracted.

Miniprep concentrations were checked by Tim:

Part DNA concentration 260/280 260/230
EP-1 75.7 2.0 2.1
EP-2 103.2 1.9 1.3
EP-3 139.0 1.8 1.4
EP-4 108.0 1.9 1.3
EP-7 73.8 1.9 1.1
EP-8 119.9 2.0 2.3
pKD3 30.4 2.2 1.3
pKD4 36.2 2.0 1.0
pKD46 90.0 1.9 1.0
RbCl-2 92.6 2.0 1.9
RbCl-4 109.6 1.9 1.4
RbCl-5 162.0 1.9 1.2
RbCl-6 79.8 2.0 1.7
RbCl-7 102.7 1.9 1.4
pTet-RBS 68.9 1.9 1.1
CA 44.6 1.9 1.1

Todays 26 well 1% gel:

Gel110809-delay-conj-linear.png


Well Part Expected Plasmid Size Status
1 EP-1 2953
2 EP-2 2915
3 EP-3 2292
4 EP-4 2894
5 EP-7 3040
6 EP-8 2374
7 RbCl-2 2915
8 RbCl-4 2894
9 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder]
10 RbCl-5 2176
11 RbCl-6 2984
12 RbCl-7 3040
13 pKD3 2804
14 pKD4 3267
15 pKD46 6329
16 pTet-RBS 2153
17 CA 4444
18 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder]

Todays 8 well 1% gel:

Gel110809excision-8well.png
Well Part Expected Plasmid Size Status
1  !E Upsteam 1114
2  !F Upsteam 913
3  !F downstream 919
4 Backbone pSB1AK3
5 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder]
6 pTet-RBS E+S Digest
7 trbK X+P Digest

Daniel

First success, got colonies in all the plates which is strange due everybody had difficulties with heat shock method which made us decide to make more electro-competent cells. Me and Sriram prepare these cells today.

From the colonies in the plates I made tube cultures by duplicate, let's wait one night for more growth.