"
Team:Tokyo Metropolitan/Notebook/A31
From 2011.igem.org
(→Speeding Up) |
|||
| Line 1: | Line 1: | ||
| - | =Speeding Up= | + | = -Speeding Up- = |
| - | + | ||
| - | + | ||
-Treat restriction enzyme | -Treat restriction enzyme | ||
| + | |||
HNS(wt) -> X,S | HNS(wt) -> X,S | ||
| + | |||
I120260 -> Bln I | I120260 -> Bln I | ||
| + | |||
for two hours. | for two hours. | ||
| + | |||
-DNA refining | -DNA refining | ||
| + | |||
Phenol/Chloroform preparation and ethanol precipitation | Phenol/Chloroform preparation and ethanol precipitation | ||
| + | |||
-Measurement of DNA concentration | -Measurement of DNA concentration | ||
| + | |||
0.04 ug/ml | 0.04 ug/ml | ||
| + | |||
-Ligation | -Ligation | ||
| + | |||
HNS(wt):I20260 (expect GFP) = 3:1 | HNS(wt):I20260 (expect GFP) = 3:1 | ||
| + | |||
-Transformation | -Transformation | ||
| Line 21: | Line 29: | ||
| - | = | + | = -Visualization- = |
| - | + | ||
| - | + | ||
-Plasmid extraction | -Plasmid extraction | ||
| + | |||
-Treat restriction enzyme (only S) | -Treat restriction enzyme (only S) | ||
| + | |||
-Electrophoresis | -Electrophoresis | ||
| + | |||
Results | Results | ||
| + | |||
Cleared plasmid extraction but retest to check for the protocol. | Cleared plasmid extraction but retest to check for the protocol. | ||
| + | |||
| + | Checked plates | ||
| + | |||
| + | Checked plates transformed after ligate one day ago. Checked colony on all plates J23100:K317037 = 1:4,1:8 and 1:0. | ||
| + | |||
| + | Treated restriction enzyme | ||
| + | |||
| + | Treated P0440 restriction enzyme with xba and pst and incubated at 37 deg C for 6 hours. | ||
| + | |||
| + | Colony PCR | ||
| + | |||
| + | Did colony PCR for 32 colony (16 colony for J23100:K317037=1:4, 14 colony for J23100:K317037=1:8 and 2 colony for only J23100). | ||
| + | |||
| + | Master plates have been incubating. | ||
| + | |||
| + | |||
| + | Electrophoresis | ||
| + | |||
| + | Checked results for colony PCR and treated restriction enzyme by electrophoresis. Showed two bands with colony PCR. Did not show bands with P0440 treated restriction enzyme. | ||
| + | |||
| + | |||
| + | Pre-cluture | ||
| + | |||
| + | Pre-clutured six tubes : three P0440 (named Mr.P, P-chan and P-ko) and colony of master plates (10,16 and 26). Liquid midum contained ampicillin. Colony of master plates used showing a band by electrophoresis. Incubated at 37 deg C after pre-cluture. | ||
Revision as of 10:00, 5 October 2011
-Speeding Up-
-Treat restriction enzyme
HNS(wt) -> X,S
I120260 -> Bln I
for two hours.
-DNA refining
Phenol/Chloroform preparation and ethanol precipitation
-Measurement of DNA concentration
0.04 ug/ml
-Ligation
HNS(wt):I20260 (expect GFP) = 3:1
-Transformation
Transformed DAN solution.
-Visualization-
-Plasmid extraction
-Treat restriction enzyme (only S)
-Electrophoresis
Results
Cleared plasmid extraction but retest to check for the protocol.
Checked plates
Checked plates transformed after ligate one day ago. Checked colony on all plates J23100:K317037 = 1:4,1:8 and 1:0.
Treated restriction enzyme
Treated P0440 restriction enzyme with xba and pst and incubated at 37 deg C for 6 hours.
Colony PCR
Did colony PCR for 32 colony (16 colony for J23100:K317037=1:4, 14 colony for J23100:K317037=1:8 and 2 colony for only J23100).
Master plates have been incubating.
Electrophoresis
Checked results for colony PCR and treated restriction enzyme by electrophoresis. Showed two bands with colony PCR. Did not show bands with P0440 treated restriction enzyme.
Pre-cluture
Pre-clutured six tubes : three P0440 (named Mr.P, P-chan and P-ko) and colony of master plates (10,16 and 26). Liquid midum contained ampicillin. Colony of master plates used showing a band by electrophoresis. Incubated at 37 deg C after pre-cluture.
