"
Team:Tsinghua/Notebook/7 August 2010
From 2010.igem.org
(→Module I, DT and Fan's part:) |
(→Module I, group 2(b)) |
||
| Line 54: | Line 54: | ||
Transform the ligation product into bacterial cells immediately. Spread about 200μl of the resulting solutions on LB plates (with 0.1% ampicillin). | Transform the ligation product into bacterial cells immediately. Spread about 200μl of the resulting solutions on LB plates (with 0.1% ampicillin). | ||
| + | == Module I, group 2c == | ||
| + | Considering some special requirements and limitation of available resources, we changed primers and plasmid for the constructs. Morever, we come to use SuperMix from Transgen as our PCR npolymerase. | ||
| + | ---- | ||
| + | |||
| + | PCR system (supermix): | ||
| + | |||
| + | Supermix 25ul | ||
| + | H2O 21ul | ||
| + | template 2ul | ||
| + | primer(up) 1ul | ||
| + | primer(down) 1ul | ||
| + | Total 50ul | ||
| + | ---- | ||
| + | |||
| + | PCR program | ||
| + | 1.94°C 5min | ||
| + | 2.94°C 30s | ||
| + | 3.50-60°C(Grad) 30s | ||
| + | 4.72°C 1min | ||
| + | cycle X 30, from 2 to 4 | ||
| + | ---- | ||
| + | Result: the result is really exciting! Specific target genes are successfully modified and purified for further experiment. | ||
Revision as of 18:35, 26 October 2010
Module I, group 2(b)
Double digestion of the eGFP, chlr and pUC19 for each pair of restriction sites. Digest at 37℃.
Double digestion system:
eGFP:
H2O 2μl buffer Tango 4μl eGFP 10μl SalI 2μl HindIII 2μl Total 20μl
pUC19 for E:
H2O 10μl buffer Tango 4μl pUC19 2μl SalI 2μl HindIII 2μl Total 20μl
chlr:
H2O 5μl buffer Tango 2μl chlr 10μl KpnI 3μl Total 20μl 37℃ 1h, then + buffer Tango 2.5μl + EcoRI 1μl Total 23.5μl
pUC19 for C:
H2O 13μl buffer Tango 2μl pUC19 2μl KpnI 3μl Total 20μl 37℃ 1h, then + buffer Tango 2.5μl + EcoRI 1μl Total 23.5μl
Productions purify and measure the concentration. Ligate with NEB T4 ligase at 25℃ for 1h.
Ligation system:
H2O 1.2μl 10×buffer 1μl fragment 2.3μl plasmid 5μl T4 ligase 0.5μl Total 10μl
Transform the ligation product into bacterial cells immediately. Spread about 200μl of the resulting solutions on LB plates (with 0.1% ampicillin).
Module I, group 2c
Considering some special requirements and limitation of available resources, we changed primers and plasmid for the constructs. Morever, we come to use SuperMix from Transgen as our PCR npolymerase.
PCR system (supermix):
Supermix 25ul H2O 21ul template 2ul primer(up) 1ul primer(down) 1ul Total 50ul
PCR program 1.94°C 5min 2.94°C 30s 3.50-60°C(Grad) 30s 4.72°C 1min cycle X 30, from 2 to 4
Result: the result is really exciting! Specific target genes are successfully modified and purified for further experiment.
