Team:TUDelft/11 August 2009

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='''11 August 2009'''=
='''11 August 2009'''=
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{{Template:TUDelftiGEM2009_end}}
 
===Tim Weenink===
===Tim Weenink===
imaged the gel i ran yesterday:
imaged the gel i ran yesterday:
 +
 +
[[Image:Tim110809!B!C!S.png|thumb|center|450px]]
 +
 +
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:
 +
<br>
 +
===Sriram===
 +
Today I did the miniprep of the assemblies cultured yesterday. The concentrations are given in Calin's section. Also I did restriction of the DNA samples and the gel was run. The image of it is given below with Calin's section.
 +
===Calin===
 +
 +
Growth in all tubes, miniprep was done.
 +
 +
Growth on all plates.
 +
 +
Checked concentrations from yesterday:
 +
 +
{| border="1" align="center"
 +
| Part || DNA concentration
 +
|- align="center"
 +
| trbK || 1.9
 +
|- align="center"
 +
| pSB1A3 || 5.2
 +
|- align="center"
 +
| pSB4A5 || 5.2
 +
|- align="center"
 +
| pSB1C3 || 3.5
 +
|- align="center"
 +
| GFP-gen || 1.9
 +
|- align="center"
 +
| 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:
 +
 +
{| border="1" align="center"
 +
| Part || DNA concentration || 260/280 || 260/230
 +
|- align="center"
 +
| EP-1 || 75.7 || 2.0 || 2.1
 +
|- align="center"
 +
| EP-2 || 103.2 || 1.9 || 1.3
 +
|- align="center"
 +
| EP-3 || 139.0 || 1.8 || 1.4
 +
|- align="center"
 +
| EP-4 || 108.0 || 1.9 || 1.3
 +
|- align="center"
 +
| EP-7 || 73.8 || 1.9 || 1.1
 +
|- align="center"
 +
| EP-8 || 119.9 || 2.0 || 2.3
 +
|- align="center"
 +
| pKD3 || 30.4 || 2.2 || 1.3
 +
|- align="center"
 +
| pKD4 || 36.2 || 2.0 || 1.0
 +
|- align="center"
 +
| pKD46 || 90.0 || 1.9 || 1.0
 +
|- align="center"
 +
| RbCl-2 || 92.6 || 2.0 || 1.9
 +
|- align="center"
 +
| RbCl-4 || 109.6 || 1.9 || 1.4
 +
|- align="center"
 +
| RbCl-5 || 162.0 || 1.9 || 1.2
 +
|- align="center"
 +
| RbCl-6 || 79.8 || 2.0 || 1.7
 +
|- align="center"
 +
| RbCl-7 || 102.7 || 1.9 || 1.4
 +
|- align="center"
 +
| pTet-RBS || 68.9 || 1.9 || 1.1
 +
|- align="center"
 +
| CA || 44.6 || 1.9 || 1.1
 +
|}
 +
 +
Todays 26 well 1% gel:
 +
 +
[[Image:Gel110809-delay-conj-linear.png|thumb|center|450px]]
 +
 +
 +
{| border="1" align="center"
 +
| Well || Part || Expected Plasmid Size || Status
 +
|- align="center"
 +
| 1 || EP-1 || 2953 || <font color=red>&#10006;</font>
 +
|- align="center"
 +
| 2 || EP-2 || 2915 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 3 || EP-3 || 2292 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 4 || EP-4 || 2894 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 5 || EP-7 || 3040 || <font color=red>&#10006;</font>
 +
|- align="center"
 +
| 6 || EP-8 || 2374 || <font color=red>&#10006;</font>
 +
|- align="center"
 +
| 7 || RbCl-2 || 2915 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 8 || RbCl-4 || 2894 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 9 || [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ||  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 10 || RbCl-5 || 2176 || <font color=red>&#10006;</font>
 +
|- align="center"
 +
| 11 || RbCl-6 || 2984 || <font color=red>&#10006;</font>
 +
|- align="center"
 +
| 12 || RbCl-7 || 3040 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 13 || pKD3 || 2804 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 14 || pKD4 || 3267 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 15 || pKD46 || 6329 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 16 || pTet-RBS || 2153 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 17 || CA || 4444 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 18 || [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ||  || <font color=limegreen>&#10004;</font>
 +
|}
 +
 +
Todays 8 well 1% gel:
 +
 +
[[Image:Gel110809excision-8well.png|thumb|center|250px]]
 +
 +
{| border="1" align="center"
 +
| Well || Part || Expected Plasmid Size || Status
 +
|- align="center"
 +
| 1 || !E Upsteam || 1114 || <font color=red>&#10006;</font>
 +
|- align="center"
 +
| 2 || !F Upsteam || 913 || <font color=red>&#10006;</font>
 +
|- align="center"
 +
| 3 || !F downstream || 919 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 4 || Backbone pSB1AK3 ||  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 5 || [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ||  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 6 || pTet-RBS E+S Digest ||  ||
 +
|- align="center"
 +
| 7 || trbK X+P Digest ||  || <font color=limegreen>&#10004;</font>
 +
|}
 +
 +
===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.
 +
 +
{{Template:TUDelftiGEM2009_end}}

Latest revision as of 08:22, 20 October 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:

Sriram

Today I did the miniprep of the assemblies cultured yesterday. The concentrations are given in Calin's section. Also I did restriction of the DNA samples and the gel was run. The image of it is given below with Calin's section.

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.

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