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EPF-Lausanne/25 August 2009
From 2009.igem.org
Contents |
Wet Lab
Miniprep:
Made according to the protocol of Aisgen Miniprep Kit Elution in 50 ul. The RD2 #5 plasmids prep as some that have already been prepared will be sent for sequencing this afternoon.
Mediums
New medium were made to grow the double-transformed DH5 a (LovTap BB and RO2) -> should be without thy.
M9 was redone, pH adjusted with amino acid and filtred. for 1L:
- 5g glucose
- 6g Na2PO4
- 3g KH2PO4
- 1g NH4Cl
- 0.5g NaCl
- 0.12g MgSO4
- 0.01g CaCl2
Trp Operon synthesis
Using the Klenow fragments protocol and primers:
- Trp operon Rev
- Trp operon Fwd
| 2x Trp | 1x Negative Control | |
|---|---|---|
|
NEB 2 (10x) |
2ul each |
2ul |
|
dNTP |
1ul each |
1ul each |
|
template primers |
1ul (RV) + 1.5ul (FW) each |
- |
|
Klenow enzyme |
1 ul each |
1 ul |
|
dH2O |
18.5 ul each |
21ul |
|
total |
25 ul each |
25 ul |
The primers volumes were directly taken from the original primer dilution (iGEM 09 primers box)
1. Running Klenow 1 file on thermal cycler to denature and slowlyy anneal primers (self-annealing) 2. Running Klenow file after adding the Klenow enzyme (for extension)
DNA preparation
For sending to sequencing
| sample name | init concentration [ng/ul] | final volume [ul] | final concentration [ng/ul] | sample [ul] | H20 |
|---|---|---|---|---|---|
|
BB1 |
180.5 |
50 |
25 |
6.94 |
43.06 |
|
BB3 |
215.5 |
50 |
25 |
5.8 |
44.2 |
|
BB5 |
350.5 |
50 |
25 |
3.57 |
46.43 |
|
BB6 |
142.5 |
50 |
25 |
8.77 |
41.23 |
|
RD2 #5 |
81.5 |
50 |
25 |
15.34 |
34.66 |
|
LTT=LT5 |
326 |
50 |
25 |
3.83 |
46.17 |
|
LR2 |
92 |
50 |
25 |
13.59 |
36.41 |
|
LR5 |
47.5 |
50 |
25 |
26.32 |
23.68 |
|
LR7 |
113.5 |
50 |
25 |
11 |
39 |
|
RD2 #8 |
247 |
50 |
25 |
5.06 |
46.94 |
|
RD2 #4 |
226 |
50 |
25 |
5.53 |
46.47 |
|
RD2 #10 |
200 |
50 |
25 |
6.25 |
43.75 |
|
LT2 |
141 |
50 |
25 |
8.87 |
41.13 |
|
LT6 |
152 |
50 |
25 |
8.22 |
41.78 |
|
LT12 |
142 |
50 |
25 |
8.8 |
41.2 |
Note : BBx means LovTAP biobrick. We identified 7 clones that worked, so x is the number of the clone. RO2#x : we have 4 clones that worked for the read out 2 but the RO2#8 seems to have a weird behaviour even if we know it has the insert, so we didn't use it afterwards.
Double transformation
We made a double transformation of RO2#4/BB1, RO2#5/BB3, RO2#8/BB5 and RO2#10/BB6.
| RO2# [ng/ul] | BB [ng/ul] | RO2# [ng/ul] | BB [ng/ul] | |
|---|---|---|---|---|
|
RO2#4 / BB1 |
226 |
180.5 |
4.45 |
5.55 |
|
RO2#5 / BB3 |
215 |
215.5 |
5 |
5 |
|
RO2#8 / BB5 |
247 |
350.5 |
5.86 |
4.14 |
|
RO2#10 / BB6 |
200 |
142.5 |
4.16 |
5.84 |
We had each time a total volume of 10ul of DNA, with the same amount of each DNA. The standard protocol was used and the cells were then spread over plates containing Kana + Amp as antibiotics.
People in the lab
Basile, Rafael, Nicolas
