DIGESTION
- BBa_E0840
- BBa_R0082
- BBa_M30109
For the first composite between the E0840 and R0082 parts, as R0082 was chosen as the backbone and E0840 the downstream reporter.
- R0082 was digested with P and S
- To 30ul of R0082 we added
- 0.5ul, 100xBSA
- 5ul, buffer 2
- 1μL, PstI
- 1μL, SpeI
- 12.5μL, H2O
- The 50μL reaction was placed at 37C for 2 hrs
- E0840 was digested with P and X
- To 30ul of E0840 we added
- 0.5ul, 100xBSA
- 5ul, buffer 3
- 1μL, PstI
- 1μL, XbaI
- 12.5μL, H2O
- The 50μL reaction was placed at 37C for 2 hrs.
Gel preperation
prepared 1% and 2% agarose gel
1% gel, loading
- 1Kb ladder
- 50ul, R0082, P/S
- 5μL, R0082, P/S
- Blank
- 5μL, E0840, P/X
- Blank
- Blank
- Blank
2% gel, loading
- 100 bp ladder
- 50ul, E0840, P/X
- 5μL, E0840, P/X
- Blank
- Blank
- Blank
- Blank
- Blank
Gel purification
the bands were cut with scapel from the gels.
- E0840, 900bp
- R0082, ~3kb
the method, book
Ligation
To ligate the cognate promnoter R0082 with reporter,E0840.
- Add 1μL of R0082 backbone
- Add 7μL of E0840 insert
- Add 1μL of ligase buffer
- Add 1μL of ligase
- Incubate at room temp for 2 hrs
Transformation
- To 50μL of competent cell (XL-1 Blue)
- Add 10μL of ligase reaction
- Place for 5 min on ice
- Place in the 42°C waterbath for 1 min
- Put back on ice for 5 min
- Add 250μL Soc
- Place in the shaker for 1 hr
- Plate on agar with appropriate antibiotic (Ampicillin) using glass beads
- Put in the incubater at 37°C
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