"
Team:Peking/Notebook/XTeng
From 2010.igem.org
(→September) |
(→September) |
||
| (3 intermediate revisions not shown) | |||
| Line 129: | Line 129: | ||
|- | |- | ||
|} | |} | ||
| - | [ | + | [<html><a href="#top">TOP</a></html>] |
| - | + | ||
===7.5-7.11=== | ===7.5-7.11=== | ||
| Line 227: | Line 226: | ||
Re-design for MBP & DsbA-MBP construction. | Re-design for MBP & DsbA-MBP construction. | ||
| - | |||
==August== | ==August== | ||
| Line 280: | Line 278: | ||
|style="text-align:center"| - | |style="text-align:center"| - | ||
|} | |} | ||
| - | [ | + | [<html><a href="#top">TOP</a></html>] |
===8.9-8.15=== | ===8.9-8.15=== | ||
MBP-His construction | MBP-His construction | ||
| Line 384: | Line 382: | ||
|style="text-align:center"| - | |style="text-align:center"| - | ||
|} | |} | ||
| - | [ | + | [<html><a href="#top">TOP</a></html>] |
| - | + | ||
===9.6-9.12=== | ===9.6-9.12=== | ||
| Line 409: | Line 406: | ||
===9.27-10.3=== | ===9.27-10.3=== | ||
1-18I-pag, 2-2M-pag, 1-18E-pag construction | 1-18I-pag, 2-2M-pag, 1-18E-pag construction | ||
| - | |||
==October== | ==October== | ||
| Line 463: | Line 459: | ||
|style="text-align:center"| - | |style="text-align:center"| - | ||
|} | |} | ||
| - | [ | + | [<html><a href="#top">TOP</a></html>] |
| - | + | ||
===10.4-10.10=== | ===10.4-10.10=== | ||
western blot for mercury Lpp-OmpA-MBP | western blot for mercury Lpp-OmpA-MBP | ||
Latest revision as of 09:58, 27 October 2010
NOTEBOOK HOME
CountDown
Vistors From...
Xin Teng's Notes
There are many pictures and graphs in my notes, I strongly recommand you to download them for more precise reading.
Contents
June
6.7-6.13
Primer design
- For expression test
pET21a-MBP-His
pET 39b DsbA-MBP
pMAL p4X-MBP-MBP
pET21a-MerR-His
pET 39b DsbA-MerR
pMAL p4X-MBP-MerR
- For standard part
MBP_SD
DsbA-MBP_SD
MBP-MBP_SD
MerR_SD
DsbA-MerR_SD
MBP-MerR_SD
6.14-6.20
Literature Review & Experimental Design for Periplasmic MBP
July
| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
| - | - | - | - | 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 |
[TOP]
7.5-7.11
DsbA-MerR (Periplasmic MerR) construction
Successful clones have been verified by sequencing.
Standardization for DsbA-MerR
Double digest shows the right result. Need to be sequenced.
MBP-His (cytosol MBP for expression test) construction
Double digest result shows that clone have failed. No band of right molecular weight can be detected.
MBP (cytosol MBP for mercury binding test) construction
Clones have been constructed, but need to be verified by double digestion.
Mercury Semiquantative Detection
Solution Preparation
1 g/L Dithizone Cloroform solution
1.038×10-3 mol/L HgCl2
Dithizone Eluant
Na2S detoxic solution
1 mol/L NaOH
Concentrated HNO3
Standard Mercury Concentration Determination
Mercury concentrated 25 times
Color change can be easily seen, but out of linear range of the standard curve.
7.12-7.18
DsbA-MBP (periplasmic MBP for expression test) construction
Band of expected molecular weight can be detected by double digest. Need to be sequenced.
Re-design the construction strategy for DsbA-MerR(MBP)
MBP-His & MBP construction
Failed.
Mercury Semiquantative Determination
Standard solution concentrated 10 times.
There is no significant difference between detected mercury concentration prepared in LB or water. But the absolute value is too low.
7.19-7.25
DsbA-MerR construction. Need to be verified.
DsbA-MBP construction. Need to be verified.
SDS-PAGE & Western Blot
Buffer preparation
5 × SDS-PAGE buffer
Tris – HCl pH 6.8
Tris – HCl pH 8.8
30% Acr / Bis-Acr
5 × TBS
5 × Transfer Buffer
1 × TTBS
SDS-PAGE result of DsbA-MerR
Overexpression band can be detected at the expected molecular weight (40 kD).
7.26-7.28 & 8.3-8.8
Double digest verification for DsbA-MBP, MBP-His & MBP.
Failed.
Check the template of MBP by sequencing.
Re-design for MBP & DsbA-MBP construction.
August
| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
| 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 | - | - | - | - |
[TOP]
8.9-8.15
MBP-His construction
Sequencing.
MBP construction
Sequencing.
DsbA-MBP construction
strategy I
Sequencing.
Strategy II
Sequencing.
Standardization for DsbA-MerR
Sequencing.
Standardization for DsbA-MBP
DsbA-MerR sample preparation for SDS-PAGE
8.16-8.22
DsbA-MerR standardization finished.
Re-construct DsbA-MBP
Expression condition optimization for DsbA-MerR
8.23-8.29
SDS-PAGE & Western Blot for DsbA-MerR
SDS-PAGE & Western Blot for DsbA-MBP
The ratio of the primary antigen to secondary antigen should be optimized to get better figures.
8.30-9.5
SDS-PAGE for T3 polymerase
SDS-PAGE & Western Blot for MBP
The ratio of the primary antigen to secondary antigen should be optimized to get better figures.
September
| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
| - | - | 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 | - | - | - |
[TOP]
9.6-9.12
Western Blot for MBP
Western Blot for DsbA-MBP
9.13-9.19
SDS-PAGE & Western Blot for lead DsbA-MBP
SDS-PAGE for lead MBP
The molecular weight of the overexpression bands does not meet our expectation. Check the plasmid.
Scan the spectrum of Crt EBI
9.20-9.26
Characterize the mutation of PmerT.
9.27-10.3
1-18I-pag, 2-2M-pag, 1-18E-pag construction
October
| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
| - | - | - | - | 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 | - | - | - | - |
[TOP]
10.4-10.10
western blot for mercury Lpp-OmpA-MBP
10.11-10.17
Cigarette digestion for our biokit.
Failed.
