Team:TUDelft/30 July 2009
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===Sriram=== | ===Sriram=== | ||
Prepared the chemically comepetent using both [https://2009.igem.org/Team:TUDelft/Protocols#Prepering_chemically_competant_cells_-_TSS_Buffer TSS Buffer protocol] and [https://2009.igem.org/Team:TUDelft/Protocols#Prepering_chemically_competant_cells_-_TMF_Buffer TMF Buffer protocol]. | Prepared the chemically comepetent using both [https://2009.igem.org/Team:TUDelft/Protocols#Prepering_chemically_competant_cells_-_TSS_Buffer TSS Buffer protocol] and [https://2009.igem.org/Team:TUDelft/Protocols#Prepering_chemically_competant_cells_-_TMF_Buffer TMF Buffer protocol]. | ||
+ | Altogether it must come for 200 transformations if all works. | ||
+ | Prepared the electrocompetent cells which was a bit laborious but we managed to prepare them using the protocol - to be updated | ||
===Tim Weenink=== | ===Tim Weenink=== |
Revision as of 11:30, 10 August 2009
Lab Notebook
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30 July 2009
Sriram
Prepared the chemically comepetent using both TSS Buffer protocol and TMF Buffer protocol. Altogether it must come for 200 transformations if all works. Prepared the electrocompetent cells which was a bit laborious but we managed to prepare them using the protocol - to be updated
Tim Weenink
prepared assembly mixes for !B, !C and !D constructs. Standard Ginko assembly kit was used. DNA concentrations as below (these mixtures were added to 7.5 µl of digestion mixture):
Assembly name | position | Components | volume in µl |
!B | Upstream | !A DNA | 33.0 |
H20 | 9.5 | ||
Downstream | BBa_B0015 DNA | 10.0 | |
H20 | 32.5 | ||
Backbone | pSB1AC3 DNA | 6.5 | |
H20 | 36 | ||
!C | Upstream | BBa_R0040 DNA | 10.0 |
H20 | 32.5 | ||
Downstream | *I6 DNA | 5.5 | |
H20 | 37.0 | ||
Backbone | pSB1AC3 DNA | 6.5 | |
H20 | 36 | ||
!D | Upstream | *I6 DNA | 5.5 |
H20 | 37.0 | ||
Downstream | BBa_K145201 DNA | 18.0 | |
H20 | 24.5 | ||
Backbone | pSB1AC3 DNA | 6.5 | |
H20 | 36 |
Ligated and transformed assemblies according to protocol.
15:20 Done plating the assemblies.
15:40 inoculated *S and *T cultures, four colonies each
16:55 Inoculated !A cultures from glycerole stock. Two tubes.
Calin
Plated three backbones left over from the transformation the other day. Did a digest on oriT-R with E and P. Made 3 x 5ml culture tubes for the pSB4C5 backbone which had a few colonies. Made electrocompetent cells with the [http://openwetware.org/wiki/Knight:Preparing_electrocompetent_cells Knight:Preparing electrocompetent cells] protocol. Flash froze tube in liquid nitrogen. Checked backbone concentrations:
Part | Concentration (ng/uL) |
rbs-GFP-term I | 102.6 |
rbs-GFP-term II | 113.5 |
pSB1A3 I | 89.1 |
pSB1A3 II | 83.2 |
pSB1A3 III | 91.9 |
pSB1A3 IV | 84.7 |
pSB4A5 I | 77.7 |
pSB4A5 II | 83.9 |
pSB4A5 III | 99.0 |
pSB4A5 IV | 116.1 |
pSB1C3 I | 102.3 |
pSB1C3 II | 49.4 |
pSB1C3 III | 81.5 |
pSB1C3 IV | 66.0 |
Ran gel on backbones and oriT digest. Gel left for too long.
Well | Part | Expected Plasmid Size | Status |
1 | rbs-GFP-term I | 2957 | ✔ |
2 | rbs-GFP-term II | 2957 | ✔ |
3 | pSB1A3 I | 2832 | ✔ |
4 | pSB1A3 II | 2832 | ✔ |
5 | pSB1A3 III | 2832 | ✔ |
6 | pSB1A3 IV | 2832 | ✔ |
7 | pSB4A5 I | 4070 | ✖ |
8 | pSB4A5 II | 4070 | ✖ |
9 | pSB4A5 III | 4070 | ✖ |
10 | pSB4A5 IV | 4070 | ✖ |
11 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ✔ | |
12 | pSB1C3 I | 2747 | ✔ |
13 | pSB1C3 II | 2747 | ✔ |
14 | pSB1C3 III | 2747 | ✔ |
15 | pSB1C3 IV | 2747 | ✔ |
16 | oriT E + P digest | 2079 + ~278 | ? |
17 | oriT E + P digest | 2079 + ~278 | ? |
18 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ✔ |
Tim Vos
Minipreped 4 x pSB1C3, 4 x pSB4A5, 2 x rbs-GFP, and 4 x pSB1A3. One pSB4A5 tube is incorrect, it shows RFP expression. Also loaded the gel.
Orr
Made some chemically competent cells using the TSS buffer protocol. Started making a Java program that will allow lock sequence design based on the chosen RBS, and will design the equivalent key based on the lock sequence. The program will incorporate a database with all the working RBS segments in the iGEM registry, so that the user can choose which RBS he wants to use.