August/10 August 2009
From 2009.igem.org
1.Min prep
Result of Nanodrop concentration check
(sample No.) (concentration) 1-6O 35.2 ng/uL 1-18L 41.8 ng/uL 1-10K 39.1 ng/uL
2.make competent cells(with CaCl2)
3.digestion and ligation
digestion with restriction enzyme
Vector1 | Insert1 | ||
---|---|---|---|
1-23L | 12 | 2-16H | 6 |
EcoRI | 1 | EcoRI | 1 |
SpeI | 1 | XbaI | 1 |
No.2 Buffer | 2 | No.2 | 2 |
dH2O | 4 | dH2O | 10 |
total | 20uL | total | 20uL |
Vector2 | Insert2 | ||
---|---|---|---|
1-23L | 12 | 3-18O | 5 |
EcoRI | 1 | EcoRI | 1 |
SpeI | 1 | XbaI | 1 |
No.2 Buffer | 2 | No.2 | 2 |
dH2O | 4 | dH2O | 11 |
total | 20uL | total | 20uL |
vector1 insert1 vector2 insert2 ladder
failure
digestion with restriction enzyme insert : E+S vector : E+x
4.Transformation
to get new plasmid
1-23L A 1-18C A 1-20F K 1-12H K 1-6I A 2-6P A 1-15N A used '8/10 competent cell