Team:TUDelft/23 July 2009

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='''23 July 2009'''=
===Tim Weenink===
===Tim Weenink===
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===Sriram===
===Sriram===
-
RThe gel was run for another 1 hour and 10 minutes and the TBE buffer was stained with 20&mico;l of Green gel stain and the bands were viewed to check whether we have the right biobricks. By this we confirmed that all the amplified biobricks to be used in Delay were extracted well and they are pure. [The gel image will be uploaded tomorrow.]
+
The agarose gel electrophoresis was run for another 1 hour and 10 minutes at 110 V. Then the gel was immersed in TBE buffer stained with 20µl of Safe Green gel stain for 1 hour and the bands were viewed to check whether we have the right biobricks. By this we confirmed that all the amplified biobricks to be used in Delay were extracted well and they are pure.
 +
 
 +
[[Image:Gel22072009-delay-conj.png‎|center|thumb|450px]]
 +
 
 +
 
 +
{| border="1" align="center"
 +
| Well || Part || Expected Plasmid Size || Status
 +
|- align="center"
 +
| 1 || [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ||  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 2 || BBa_C0040 TetR || 2739  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 3 || BBa_ E0040 GFP || 2799  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 4 || BBa_E1010 mRFPI || 5106
 +
|- align="center"
 +
| 5 || BBa_I12006 pLambclin || 4507
 +
|- align="center"
 +
| 6 || BBa_C0051 cI || 2829  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 7 || BBa_J23008 key3c || 2450  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 8 || BBa_I12006 pLambclin star || 4507
 +
|- align="center"
 +
| 9 || BBa_B0015 Double Terminator || 3318  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 10 || BBa_R0010 pLacI || 2279  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 11 || BBa_K081013 RBS-cI-RBS || 2898  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 12 || BBa_J23031 lock3c || 2398  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 13 || BBa_R0040 pTet || 2133  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 14 || BBa_B0034 RBS || 2091  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 15 || BBa_I714031 OriT-R || 2357  || <font color=red>&#10006;</font>
 +
|- align="center"
 +
| 16 || BBa_E0840 GFP generator || 2957  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 17 || BBa_J23100 strong promoter || 2948  || <font color=red>&#10006;</font>
 +
|- align="center"
 +
| 18 || BBa_I13522 pTet GFP || 3016  || <font color=limegreen>&#10004;</font>
 +
|}
 +
 
 +
 
 +
For preparing glycerol stocks and plasmid DNA extraction the 7 biobrick colonies [R0010, R0040, J23008, J23031, B0034, B0015, K081013] from the LB agar plates and 2 composite biobricks [S03335, S03473] received from iGEM HQ were cultured in 5 ml tubes with 1xAmp. The 7 biobricks were recultured since we anticipate that we may need more DNA for parts like RBS, Double terminator, etc. in more assemblies and also glycerol stocks were not made for these biobricks.
 +
 
 +
The 2 composite biobricks [S03335, S03473] were also streaked in LB Agar plates with 1xAmp for future use.
 +
 
 +
===Orr===
 +
Prepared TBE and LB Agar mediums using the standard protocols.
 +
 
 +
Looked at the negative feedback Matlab code in an attempt to write a new code that will enable us to change two variables at a time and check a set of values to find the optimal value for each parameter.
 +
 
-
For preparing glycerol stocks and plasmid DNA extraction the 7 biobrick colonies from the LB agar plates and 2 composite biobricks received from iGEM HQ were cultured in 5 ml tubes with 1xAmp. The 7 biobricks were recultured since we anticipate we may need more DNA for assembly for parts like RBS, Double terminator, etc. and also glycerol stocks were not made for these biobricks.
+
===Calin===
 +
Gel showed that oriT and promoter miniprep didnt go right. Cultured oriT, promoter, GFP gen in 5 mL tubes. Made 2 GFP plates, an RFP plate, and one with both. Plated parts from MIT (BBa_J23015, BBa_J23055). Added composite part with GFP and oriT to sandbox. Acquired some 20% L-arabinose. Looked into the proofreading PCR kit in the lab for possible use in the knockout (Roche Expand High Fidelity PCR System).
-
The 2 composite biobricks were also streaked in LB Agar plates with 1xAmp for future use.
 
{{Template:TUDelftiGEM2009_end}}
{{Template:TUDelftiGEM2009_end}}

Latest revision as of 10:28, 12 October 2009

Lab Notebook

July
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6 7 8 9 10 11 12
13 14 15 16 17 18 19
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27 28 29 30 31
August
MTWTFSS
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10 11 12 13 14 15 16
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31
September
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October
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12 13 14 15 16 17 18
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26 27 28 29 30 31
 

23 July 2009

Tim Weenink

Details of the following to be provided tomorrow:

grew up *S in LC

looked at gel

Did !A assembly again with double the amount of backbone (and rest from fridge). Did it with both DH5alpha and homemade top10 chemically competent cells, to compare transformation efficiencies. Also the right concentration of K was used

PCR'd 8 colonies from my transformation plates with VF2 and I-SceI Reverse primers to see if there was incorporation of the I-SceI restriction site

isolated plasmid from *S

Sriram

The agarose gel electrophoresis was run for another 1 hour and 10 minutes at 110 V. Then the gel was immersed in TBE buffer stained with 20µl of Safe Green gel stain for 1 hour and the bands were viewed to check whether we have the right biobricks. By this we confirmed that all the amplified biobricks to be used in Delay were extracted well and they are pure.

Gel22072009-delay-conj.png


Well Part Expected Plasmid Size Status
1 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ✔
2 BBa_C0040 TetR 2739 ✔
3 BBa_ E0040 GFP 2799 ✔
4 BBa_E1010 mRFPI 5106
5 BBa_I12006 pLambclin 4507
6 BBa_C0051 cI 2829 ✔
7 BBa_J23008 key3c 2450 ✔
8 BBa_I12006 pLambclin star 4507
9 BBa_B0015 Double Terminator 3318 ✔
10 BBa_R0010 pLacI 2279 ✔
11 BBa_K081013 RBS-cI-RBS 2898 ✔
12 BBa_J23031 lock3c 2398 ✔
13 BBa_R0040 pTet 2133 ✔
14 BBa_B0034 RBS 2091 ✔
15 BBa_I714031 OriT-R 2357 ✖
16 BBa_E0840 GFP generator 2957 ✔
17 BBa_J23100 strong promoter 2948 ✖
18 BBa_I13522 pTet GFP 3016 ✔


For preparing glycerol stocks and plasmid DNA extraction the 7 biobrick colonies [R0010, R0040, J23008, J23031, B0034, B0015, K081013] from the LB agar plates and 2 composite biobricks [S03335, S03473] received from iGEM HQ were cultured in 5 ml tubes with 1xAmp. The 7 biobricks were recultured since we anticipate that we may need more DNA for parts like RBS, Double terminator, etc. in more assemblies and also glycerol stocks were not made for these biobricks.

The 2 composite biobricks [S03335, S03473] were also streaked in LB Agar plates with 1xAmp for future use.

Orr

Prepared TBE and LB Agar mediums using the standard protocols.

Looked at the negative feedback Matlab code in an attempt to write a new code that will enable us to change two variables at a time and check a set of values to find the optimal value for each parameter.


Calin

Gel showed that oriT and promoter miniprep didnt go right. Cultured oriT, promoter, GFP gen in 5 mL tubes. Made 2 GFP plates, an RFP plate, and one with both. Plated parts from MIT (BBa_J23015, BBa_J23055). Added composite part with GFP and oriT to sandbox. Acquired some 20% L-arabinose. Looked into the proofreading PCR kit in the lab for possible use in the knockout (Roche Expand High Fidelity PCR System).

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