Team:DTU-Denmark/Notebook/20 August 2013
From 2013.igem.org
(→PCR to linearize pZA21::RFP::araBAD) |
(→Lab 208) |
||
(9 intermediate revisions not shown) | |||
Line 1: | Line 1: | ||
{{:Team:DTU-Denmark/Templates/StartPage|20 August 2013}} | {{:Team:DTU-Denmark/Templates/StartPage|20 August 2013}} | ||
- | |||
Navigate to the [[Team:DTU-Denmark/Notebook/19_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/21_August_2013|Next]] Entry | Navigate to the [[Team:DTU-Denmark/Notebook/19_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/21_August_2013|Next]] Entry | ||
- | + | =Lab 208= | |
- | = | + | |
<hr/> | <hr/> | ||
==Main purpose== | ==Main purpose== | ||
Line 92: | Line 90: | ||
* 1 kb ladder | * 1 kb ladder | ||
- | + | [[File:2013-08-20 screen big.jpg|600px]] | |
* 1 kb ladder | * 1 kb ladder | ||
Line 110: | Line 108: | ||
* 1 kb ladder | * 1 kb ladder | ||
- | + | [[File:2013-08-20 screen small.jpg|600px]] | |
+ | |||
+ | gel on today's PCR to linearize pZA21::ara | ||
+ | |||
+ | * 1 kb ladder | ||
+ | * sample 1 | ||
+ | * sample 2 | ||
+ | * sample 3 | ||
+ | * sample 4 | ||
+ | * negative controll | ||
+ | * 1 kb ladder | ||
+ | |||
+ | [[File:2013-08-20 test gel pza.jpg|600px]] | ||
+ | |||
+ | Two samples were lost because the lids opened during the PCR reaction and sample evaporated. | ||
===purification gels=== | ===purification gels=== | ||
- | Pooled Nir2 and Nir1 samples that gave bands | + | Pooled Nir2 and Nir1 samples for Morten Nørholm that gave bands. |
* 1 kb ladder | * 1 kb ladder | ||
Line 128: | Line 140: | ||
* Nir1 | * Nir1 | ||
* 1 kb ladder | * 1 kb ladder | ||
+ | |||
+ | [[File:2013-08-20 puri lin pza21ara nir2 nir1.jpg|600px]] | ||
Second purification | Second purification | ||
Line 139: | Line 153: | ||
* Nir2 | * Nir2 | ||
* 1 kb ladder | * 1 kb ladder | ||
+ | |||
+ | [[File:2013-08-20 puri2 pza nir.jpg|600px]] | ||
==Conclusion== | ==Conclusion== | ||
<hr/> | <hr/> | ||
+ | |||
+ | Something is definitely wrong with our gels. It seems the gradient PCR did not give the right product. Amplification of the linearized pZA21::ara worked. | ||
+ | |||
Navigate to the [[Team:DTU-Denmark/Notebook/19_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/21_August_2013|Next]] Entry | Navigate to the [[Team:DTU-Denmark/Notebook/19_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/21_August_2013|Next]] Entry | ||
{{:Team:DTU-Denmark/Templates/EndPage}} | {{:Team:DTU-Denmark/Templates/EndPage}} |
Latest revision as of 17:46, 28 September 2013
20 August 2013
Contents |
Lab 208
Main purpose
- PCR to linearize pZA21::RFP::araBAD
Who was in the lab
Kristian, Helen, Henrike, Julia
Procedure
PCR to linearize pZA21::RFP::araBAD
- Template: pZA21::RFP::araBAD
- Primers: 55a and 55b
- Program:
temperature | time | cycles |
---|---|---|
98C | 2:00 | - |
98C | 0:10 | 36 |
65C | 1:00 | 36 |
72C | 3:00 | 36 |
72C | 5:00 | - |
10C | hold | - |
The table shows the composition of each reaction:
component (per reaction) | 1 | 2 | 3 | 4 | 5 | 6 | Neg |
---|---|---|---|---|---|---|---|
dNTPs | 1uL | 1uL | 1uL | 1uL | 1uL | 1uL | - |
buffer (GC/HF + volume) | HF + 10uL | HF + 10uL | HF + 10uL | GC + 10uL | HF + 10uL | HF + 10uL | HF + 10uL |
X7 polymerase | 0.5uL | 0.5uL | 0.5uL | 0.5uL | 0.5uL | 0.5uL | 0.5uL |
MilliQ water | 29uL | 21.5uL | 31.5uL | 29uL | 31.5uL | 31.5uL | 32.5uL |
template | 1uL | 1uL | 1uL | 1uL | 1uL | 1uL | - |
FW primer 55a | 3uL | 3uL | 3uL | 3uL | 3uL | 3uL | 3uL |
RV primer 55b | 3uL | 3uL | 3uL | 3uL | 3uL | 3uL | 3uL |
DMSO (100%) | 2.5uL (5%) | - | - | 2.5uL (5%) | - | - | - |
Betaine (5M) | - | 10uL (1M) | - | - | - | - | - |
Repeated reaction mix number 1 and ran on same program to get more backbone.
Results
Gels
loaded more samples from the screening PCR for BioBrick creation yesterday
- 1 kb ladder
- screening D12
- screening D11
- screening D10
- screening D9
- screening D8
- screening D7
- screening D6
- screening D5
- screening D4
- screening D3
- screening D2
- screening D1
- screening C12
- screening C11
- screening C10
- screening C9
- screening C8
- 1 kb ladder
- 1 kb ladder
- screening C7
- screening C6
- screening C5
- screening C4
- screening C3
- screening C2
- screening C1
- screening B12
- screening B11
- screening B10
- screening B9
- screening B8
- screening B7
- 1 kb ladder
gel on today's PCR to linearize pZA21::ara
- 1 kb ladder
- sample 1
- sample 2
- sample 3
- sample 4
- negative controll
- 1 kb ladder
Two samples were lost because the lids opened during the PCR reaction and sample evaporated.
purification gels
Pooled Nir2 and Nir1 samples for Morten Nørholm that gave bands.
- 1 kb ladder
- pZA21::RFP::AraBAD
- pZA21::RFP::AraBAD
- pZA21::RFP::AraBAD
- Nir2
- Nir2
- Nir2
- Nir1
- Nir1
- Nir1
- Nir1
- 1 kb ladder
Second purification
- 1 kb ladder
- pZA21::RFP::AraBAD, sample 1 (from todays PCR)
- Nir1
- Nir1
- Nir2
- Nir2
- Nir2
- 1 kb ladder
Conclusion
Something is definitely wrong with our gels. It seems the gradient PCR did not give the right product. Amplification of the linearized pZA21::ara worked.
Navigate to the Previous or the Next Entry