Team:TUDelft/18 August 2009

From 2009.igem.org

Lab Notebook

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August
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October
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18 August 2009

Sriram

Checking the yesterday's gel (shown below) I found that since the ladder didn't run well we couldn't interpret the size properly. hence I decided to do the whole assembly again. Or else we need to follow the back up plan for negative cascade alone created by Daniel. So today to start everything again I made cultures for all the primary biobricks to start the assembly all over again. We also have one problem the restriction enzyme SpeI is getting over. So it was ordered today.

Calin

Yesterdays 20 well 1% gel:

Gel17082009-20well-1pc-sd-c.png


Well Part Expected Plasmid Size Status
1
2
3
4
5
6 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ✔
7 Colony PCR 1A - R751ΔtrbK 1616 ✖
8 Colony PCR 1A - R751ΔtrbK 1616 ✖
9 Colony PCR R751 + pKD46 + trbK_KO_mod_S 348 ✔
10 Colony PCR R751ΔoriTR -L-ara +PCR control  ?
11 Colony PCR R751 + pKD46 + oriTR_KO_mod_S 398 ✔
12 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ✔

Yesterdays 26 well 2% gel:

Gel17082009-26well-2pc-dela.png

Well Part Expected Plasmid Size Status
1 pTet
2 RBS-cI-RBS
3 pSB1C3
4 pLacI
5 RBS
6 pSB1C3
7 pTet
8 lock3c
9 pSB1C3 (not loaded well)
10 pSB1C3
11 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ✖
12 cI
13 Double Terminator
14 pSB1C3
15 λp-in
16 RBS-GFP-Double Term
17 pSB1C3
18 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ✖
19 pLacI
20 key3c
21 pSB1AK3
22 Colony PCR 1A - R751ΔtrbK 1616 ✖
23 Colony PCR 1A - R751ΔtrbK 1616 ✖
24 Colony PCR R751 + pKD46 + trbK_KO_mod_S 348 ✔
25 Colony PCR R751ΔoriTR -L-ara +PCR control  ?
26 Colony PCR R751 + pKD46 + oriTR_KO_mod_S 398 ✔


For the trbK conjugation test 24 plates were made:


Set 1
Plate ID Antibiotics Dilution
D1tr TRI 100
D2tr TRI 10-1
D3tr TRI 10-2
D4tr TRI 10-3
D5tr TRI 10-4
T1tr TRI + CAM 100
T2tr TRI + CAM 10-1
T3tr TRI + CAM 10-2
T4tr TRI + CAM 10-3
T5tr TRI + CAM 10-4
R1tr CAM 100
R2tr CAM 10-1



Set 2
Plate ID Antibiotics Dilution
D6tr TRI 100
D7tr TRI 10-1
D8tr TRI 10-2
D9tr TRI 10-3
D10tr TRI 10-4
T6tr TRI + CAM 100
T7tr TRI + CAM 10-1
T8tr TRI + CAM 10-2
T9tr TRI + CAM 10-3
T10tr TRI + CAM 10-4
R3tr CAM 100
R4tr CAM 10-1

For the oriTR conjugation test 51 plates were made:

Set 1
Plate ID Antibiotics Dilution
D1ori TRI + CAM 100
D2ori TRI + CAM 10-1
D3ori TRI + CAM 10-2
D4ori TRI + CAM 10-3
D5ori TRI + CAM 10-4
Ts1ori AMP + CAM 100
Ts2ori AMP + CAM 10-1
Ts3ori AMP + CAM 10-2
Ts4ori AMP + CAM 10-3
Ts5ori AMP + CAM 10-4
TL1ori TRI + AMP 100
TL2ori TRI + AMP 10-1
TL3ori TRI + AMP 10-2
TL4ori TRI + AMP 10-3
TL5ori TRI + AMP 10-4
R1ori AMP 100
R2ori AMP 10-1



Set 2
Plate ID Antibiotics Dilution
D6ori TRI + CAM 100
D7ori TRI + CAM 10-1
D8ori TRI + CAM 10-2
D9ori TRI + CAM 10-3
D10ori TRI + CAM 10-4
Ts6ori AMP + CAM 100
Ts7ori AMP + CAM 10-1
Ts8ori AMP + CAM 10-2
Ts9ori AMP + CAM 10-3
Ts10ori AMP + CAM 10-4
TL6ori TRI + AMP 100
TL7ori TRI + AMP 10-1
TL8ori TRI + AMP 10-2
TL9ori TRI + AMP 10-3
TL10ori TRI + AMP 10-4
R3ori AMP 100
R4ori AMP 10-1




Set 3
Plate ID Antibiotics Dilution
D11ori TRI + CAM 100
D12ori TRI + CAM 10-1
D13ori TRI + CAM 10-2
D14ori TRI + CAM 10-3
D15ori TRI + CAM 10-4
Ts11ori AMP + CAM 100
Ts12ori AMP + CAM 10-1
Ts13ori AMP + CAM 10-2
Ts14ori AMP + CAM 10-3
Ts15ori AMP + CAM 10-4
TL11ori TRI + AMP 100
TL12ori TRI + AMP 10-1
TL13ori TRI + AMP 10-2
TL14ori TRI + AMP 10-3
TL15ori TRI + AMP 10-4
R5ori AMP 100
R6ori AMP 10-1

Tim Weenink

Results of the 24h sequencing are in:

  • !BPEA1 (R) correct in reliable part of sequencing file
  • !BCEC1 (R) correct in reliable part of sequencing file
  • !CCEA1 (F) perfect
  • !CCEC2 (F) perfect
  • !SPEA1 (F) Assebly with !A and BBa_K142202. !A is not supposed to be there
  • !SPEA1 (R) Assebly with !A and BBa_K142202. !A is not supposed to be there
  • !SPEC1 (F) Failed (as expected)
  • !SPEC1 (R) Failed (as expected)
  • *I6F (F) Perfect
  • *S3 (F) Is not BBa_K142205, but in fact BBa_K142203
  • *S3 (R) Is not BBa_K142205, but in fact BBa_K142203

Daniel

Growth, miniprep:

Biobrick [DNA] ng/uL
C0051 45
P0440 48.2
R0051 22.8
I13504 70.1
Ca 28.1


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