NYMU Taipei/Lab Notes/2007 9 8

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Latest revision as of 10:31, 10 September 2007

PCR (H_INS_A)
- Template (H_INS): 1 ul
- 10x PCR buffer: 5 ul
- 2.5mM dNTP: 2 ul
- H_INSA-F (10pM/ul): 1.5 ul
- H_INSA-R (10pM/ul): 1.5 ul
- Pfu(Promega): 0.5 ul
- H2O: 38.5 ul
- Total: 50ul
PCR (H_INS_B)
- Template (H_INS): 1 ul
- 10x PCR buffer: 5 ul
- 2.5mM dNTP: 2 ul
- H_INSB-F (10pM/ul): 1.5 ul
- H_INSB-R (10pM/ul): 1.5 ul
- Pfu: 0.5 ul
- H2O: 38.5 ul
- Total: 50ul
program:PETT2
- 95°C:5-10min
- 95°C:1min(*)
- 55°C:1min(*)
- 72°C:1min(*)
- (*) 35 cycles
- 72°C:10min
- 4°C:∞
H_INS:28-30ul/each well
marker:5ul
H_INS_A

- from left to right
- well 1:empty
- well 2,3:INS_A 5
- well 4,5:INS_A 4
- well 6,7:INS_A 3
- well 8,9:INS_A 2
- well 10,11:INS_A 1
- well 12:marker
H_INS_B

- from left to right
- well 1:empty
- well 2,3:INS_B 5
- well 4,5:INS_B 4
- well 6,7:INS_B 3
- well 8,9:INS_B 2
- well 10,11:INS_B 1
- well 12:marker
QlAquick Gel Extraction Kit
- A1:0.08g
- A3:0.15g
- B1:0.12g
- B2:0.12g
- B3:0.16g
- B4:0.11g
- B5:0.17g
RE Digestion
- H_INS_A/B:11ul
- XbaI:1ul
- PstI:1ul
- 10X buffer:2ul
- 10X BSA:2ul
- ddH2O:3ul
- total:20ul
QlAquick Gel Extraction Kit
measuring the con.of H_INS_A/B with the spectrophotometer
- INS_A1:13ug/ul
- INS_B1:4ug/ul
- INS_B2:0ug/ul
- INS_B3:0ug/ul
- INS_B4:0ug/ul
- INS_B5:0ug/ul
Ligation(H_INS_A)
- H_INS_A1:4ul
- 10X ligase:1 ul
- 10X buffer:1 ul
- ddH2O:4ul
- total: 10ul
Ligation(H_INS_B)
- H_INS_B1:8ul
- 10X ligase:1 ul
- 10X buffer:1 ul
- ddH2O:0ul
- total: 10ul
ligation: room temp.:15 mins
transformation
- ligated DNA:2ul
- compenent cell:50ul
apply the cells on the agar plate(Amp)
culture plate failed due to lack of vector adding in the ligation process

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