"
Team:TUDelft/11 August 2009
From 2009.igem.org
(→Calin) |
(→Calin) |
||
| (7 intermediate revisions not shown) | |||
| Line 9: | Line 9: | ||
imaged the gel i ran yesterday: | imaged the gel i ran yesterday: | ||
| - | [[Image:Tim110809!B!C!S.png| | + | [[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. | 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: | 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=== | ===Calin=== | ||
| Line 90: | Line 91: | ||
Todays 26 well 1% gel: | Todays 26 well 1% gel: | ||
| - | [[Image:Gel110809-delay-conj-linear.png| | + | [[Image:Gel110809-delay-conj-linear.png|thumb|center|450px]] |
| Line 96: | Line 97: | ||
| Well || Part || Expected Plasmid Size || Status | | Well || Part || Expected Plasmid Size || Status | ||
|- align="center" | |- align="center" | ||
| - | | 1 || EP-1 || 2953 || | + | | 1 || EP-1 || 2953 || <font color=red>✖</font> |
|- align="center" | |- align="center" | ||
| 2 || EP-2 || 2915 || <font color=limegreen>✔</font> | | 2 || EP-2 || 2915 || <font color=limegreen>✔</font> | ||
| Line 104: | Line 105: | ||
| 4 || EP-4 || 2894 || <font color=limegreen>✔</font> | | 4 || EP-4 || 2894 || <font color=limegreen>✔</font> | ||
|- align="center" | |- align="center" | ||
| - | | 5 || EP-7 || 3040 || | + | | 5 || EP-7 || 3040 || <font color=red>✖</font> |
|- align="center" | |- align="center" | ||
| - | | 6 || EP-8 || 2374 || | + | | 6 || EP-8 || 2374 || <font color=red>✖</font> |
|- align="center" | |- align="center" | ||
| 7 || RbCl-2 || 2915 || <font color=limegreen>✔</font> | | 7 || RbCl-2 || 2915 || <font color=limegreen>✔</font> | ||
| Line 114: | Line 115: | ||
| 9 || [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] || || <font color=limegreen>✔</font> | | 9 || [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] || || <font color=limegreen>✔</font> | ||
|- align="center" | |- align="center" | ||
| - | | 10 || RbCl-5 || 2176 || | + | | 10 || RbCl-5 || 2176 || <font color=red>✖</font> |
|- align="center" | |- align="center" | ||
| - | | 11 || RbCl-6 || 2984 || | + | | 11 || RbCl-6 || 2984 || <font color=red>✖</font> |
|- align="center" | |- align="center" | ||
| 12 || RbCl-7 || 3040 || <font color=limegreen>✔</font> | | 12 || RbCl-7 || 3040 || <font color=limegreen>✔</font> | ||
| Line 135: | Line 136: | ||
Todays 8 well 1% gel: | Todays 8 well 1% gel: | ||
| - | [[Image:Gel110809excision-8well.png| | + | [[Image:Gel110809excision-8well.png|thumb|center|250px]] |
{| border="1" align="center" | {| border="1" align="center" | ||
| Well || Part || Expected Plasmid Size || Status | | Well || Part || Expected Plasmid Size || Status | ||
|- align="center" | |- align="center" | ||
| - | | 1 || | + | | 1 || !E Upsteam || 1114 || <font color=red>✖</font> |
|- align="center" | |- align="center" | ||
| - | | 2 || | + | | 2 || !F Upsteam || 913 || <font color=red>✖</font> |
|- align="center" | |- align="center" | ||
| - | | 3 || | + | | 3 || !F downstream || 919 || <font color=limegreen>✔</font> |
|- align="center" | |- align="center" | ||
| - | | 4 || | + | | 4 || Backbone pSB1AK3 || || <font color=limegreen>✔</font> |
|- align="center" | |- align="center" | ||
| 5 || [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] || || <font color=limegreen>✔</font> | | 5 || [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] || || <font color=limegreen>✔</font> | ||
| Line 154: | Line 155: | ||
| 7 || trbK X+P Digest || || <font color=limegreen>✔</font> | | 7 || trbK X+P Digest || || <font color=limegreen>✔</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}} | {{Template:TUDelftiGEM2009_end}} | ||
Latest revision as of 08:22, 20 October 2009
Lab Notebook
|
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
11 August 2009
Tim Weenink
imaged the gel i ran yesterday:
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:
| 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:
| 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.
