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Team:Tsinghua/Notebook/25 August 2010
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(Difference between revisions)
(New page: == Module I, DT and Fan's part: == Ligate PEMK with C. Using the PEMK①. Double digestion system: chlr: H2O 5μl buffer Tango 2μl chlr 10μl KpnI ...) |
(→Module I, group 2(b)) |
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| - | == Module I, | + | == Module I, group 2(b) == |
Ligate PEMK with C. Using the PEMK①. | Ligate PEMK with C. Using the PEMK①. | ||
| Line 12: | Line 12: | ||
Total 20μl | Total 20μl | ||
37℃ 1h, then | 37℃ 1h, then | ||
| - | + buffer Tango | + | + buffer Tango 2.5μl |
+ EcoRI 1μl | + EcoRI 1μl | ||
Total 23.5μl | Total 23.5μl | ||
| Line 22: | Line 22: | ||
KpnI 3μl | KpnI 3μl | ||
Total 20μl | Total 20μl | ||
| + | 37℃ 1h, then | ||
| + | + buffer Tango 2.5μl | ||
| + | + EcoRI 1μl | ||
| + | Total 23.5μl | ||
| + | |||
| + | Run a gel and cut to purify the digestion products. Measure the concentration. Ligate the PEM and PC with K. | ||
| + | |||
| + | Ligation system: | ||
| + | |||
| + | PEMK+C: | ||
| + | H2O 14.4μl | ||
| + | 10×buffer 2μl | ||
| + | C 1μl | ||
| + | PEMK 1.6μl | ||
| + | T4 ligase 1μl | ||
| + | Total 20μl | ||
| + | |||
| + | Transform the ligation product into bacterial cells immediately. Spread about 100μl of the resulting solutions on LB plates (with 0.1% ampicillin). | ||
| + | == Module I, Group 2c == | ||
| + | |||
| + | Ligation product is transformed in to DH5aα 24h after ligation. | ||
Latest revision as of 20:28, 26 October 2010
Module I, group 2(b)
Ligate PEMK with C. Using the PEMK①.
Double digestion system:
chlr:
H2O 5μl buffer Tango 2μl chlr 10μl KpnI 3μl Total 20μl 37℃ 1h, then + buffer Tango 2.5μl + EcoRI 1μl Total 23.5μl
PEMK:
H2O 3μl buffer Tango 2μl PEMK 12μl KpnI 3μl Total 20μl 37℃ 1h, then + buffer Tango 2.5μl + EcoRI 1μl Total 23.5μl
Run a gel and cut to purify the digestion products. Measure the concentration. Ligate the PEM and PC with K.
Ligation system:
PEMK+C:
H2O 14.4μl 10×buffer 2μl C 1μl PEMK 1.6μl T4 ligase 1μl Total 20μl
Transform the ligation product into bacterial cells immediately. Spread about 100μl of the resulting solutions on LB plates (with 0.1% ampicillin).
Module I, Group 2c
Ligation product is transformed in to DH5aα 24h after ligation.
