Team:TUDelft/30 July 2009

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='''30 July 2009'''=
='''30 July 2009'''=
 +
===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].
 +
Altogether it must come for 200 transformations if all works.
 +
Along with Calin I prepared the electrocompetent cells which was a bit laborious but we managed to prepare them.
===Tim Weenink===
===Tim Weenink===
prepared assembly mixes for !B, !C and !D constructs.
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):
 +
{| border="1" align="center"
 +
| Assembly name || position || Components || volume in µl
 +
|- align="center"
 +
| !B|| Upstream || !A DNA || 33.0
 +
|- align="center"
 +
| ||  || H20 || 9.5
 +
|- align="center"
 +
| || Downstream || BBa_B0015 DNA || 10.0
 +
|- align="center"
 +
| ||  || H20 || 32.5
 +
|- align="center"
 +
| || Backbone || pSB1AC3 DNA || 6.5
 +
|- align="center"
 +
| ||  || H20 || 36
 +
 +
|- align="center"
 +
| !C|| Upstream || BBa_R0040 DNA || 10.0
 +
|- align="center"
 +
| ||  || H20 || 32.5
 +
|- align="center"
 +
| || Downstream || *I6 DNA || 5.5
 +
|- align="center"
 +
| ||  || H20 || 37.0
 +
|- align="center"
 +
| || Backbone || pSB1AC3 DNA || 6.5
 +
|- align="center"
 +
| ||  || H20 || 36
 +
|- align="center"
 +
 +
| !D|| Upstream || *I6 DNA || 5.5
 +
|- align="center"
 +
| ||  || H20 || 37.0
 +
|- align="center"
 +
| || Downstream || BBa_K145201 DNA || 18.0
 +
|- align="center"
 +
| ||  || H20 || 24.5
 +
|- align="center"
 +
| || Backbone || pSB1AC3 DNA || 6.5
 +
|- align="center"
 +
| ||  || 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===
===Calin===
-
Plated three backbones left over from the transformation the other day. Did a digest on oriT-R with E and P.
+
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:
 +
 
 +
 
 +
{| border="1" align="center"
 +
| Part || Concentration (ng/uL)
 +
|- align="center"
 +
| rbs-GFP-term I || 102.6
 +
|- align="center"
 +
| rbs-GFP-term II || 113.5
 +
|- align="center"
 +
| pSB1A3 I || 89.1
 +
|- align="center"
 +
| pSB1A3 II || 83.2
 +
|- align="center"
 +
| pSB1A3 III || 91.9
 +
|- align="center"
 +
| pSB1A3 IV || 84.7
 +
|- align="center"
 +
| pSB4A5 I || 77.7
 +
|- align="center"
 +
| pSB4A5 II || 83.9
 +
|- align="center"
 +
| pSB4A5 III || 99.0
 +
|- align="center"
 +
| pSB4A5 IV || 116.1
 +
|- align="center"
 +
| pSB1C3 I || 102.3
 +
|- align="center"
 +
| pSB1C3 II || 49.4
 +
|- align="center"
 +
| pSB1C3 III || 81.5
 +
|- align="center"
 +
| pSB1C3 IV || 66.0
 +
|}
 +
 
 +
Ran gel on backbones and oriT digest. Gel left for too long.
 +
 
 +
[[Image:Gel30072009.png|thumb|center|450px]]
 +
 
 +
{| border="1" align="center"
 +
| Well || Part || Expected Plasmid Size || Status
 +
|- align="center"
 +
| 1 || rbs-GFP-term I || 2957 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 2 || rbs-GFP-term II || 2957 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 3 || pSB1A3 I || 2832 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 4 || pSB1A3 II || 2832 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 5 || pSB1A3 III || 2832 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 6 || pSB1A3 IV || 2832 || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 7 || pSB4A5 I || 4070  ||  <font color=red>&#10006;</font>
 +
|- align="center"
 +
| 8 || pSB4A5 II || 4070  ||  <font color=red>&#10006;</font>
 +
|- align="center"
 +
| 9 || pSB4A5 III || 4070  ||  <font color=red>&#10006;</font>
 +
|- align="center"
 +
| 10 || pSB4A5 IV || 4070  ||  <font color=red>&#10006;</font>
 +
|- align="center"
 +
| 11 || [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ||  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 12 || pSB1C3 I || 2747  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 13 || pSB1C3 II || 2747  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 14 || pSB1C3 III || 2747  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 15 || pSB1C3 IV || 2747  || <font color=limegreen>&#10004;</font>
 +
|- align="center"
 +
| 16 || oriT E + P digest || 2079 + ~278 || ?
 +
|- align="center"
 +
| 17 || oriT E + P digest || 2079 + ~278  || ?
 +
|- align="center"
 +
| 18 || [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ||  || <font color=limegreen>&#10004;</font>
 +
|}
===Tim Vos===
===Tim Vos===
-
Minipreped pSB4C5, pSB.
+
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.
{{Template:TUDelftiGEM2009_end}}
{{Template:TUDelftiGEM2009_end}}

Latest revision as of 10:30, 12 October 2009

Lab Notebook

July
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
 

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. Along with Calin I prepared the electrocompetent cells which was a bit laborious but we managed to prepare them.

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.

Gel30072009.png


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.


Retrieved from "http://2009.igem.org/Team:TUDelft/30_July_2009"