User:Hana Benzeer/Notebook/SGM Summer Project/2011/06/15

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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DIGESTION

 * 1) BBa_E0840
 * 2) BBa_R0082
 * 3) BBa_M30109

For the first composite between the E0840 and R0082 parts, as R0082 was chosen as the backbone and E0840 the downstream reporter.


 * 1) R0082 was digested with P and S
 * 2) To 30ul of R0082 we added
 * 3) 0.5ul, 100xBSA
 * 4) 5ul, buffer 2
 * 5) 1μL, PstI
 * 6) 1μL, SpeI
 * 7) 12.5μL, H2O
 * 8) The 50μL reaction was placed at 37C for 2 hrs
 * 9) E0840 was digested with P and X
 * 10) To 30ul of E0840 we added
 * 11) 0.5ul, 100xBSA
 * 12) 5ul, buffer 3
 * 13) 1μL, PstI
 * 14) 1μL, XbaI
 * 15) 12.5μL, H2O
 * 16) The 50μL reaction was placed at 37C for 2 hrs.

Gel preperation
prepared 1% and 2% agarose gel

1% gel, loading

 * 1) 1Kb ladder
 * 2) 50ul, R0082, P/S
 * 3) 5μL, R0082, P/S
 * 4) Blank
 * 5) 5μL, E0840, P/X
 * 6) Blank
 * 7) Blank
 * 8) Blank

2% gel, loading

 * 1) 100 bp ladder
 * 2) 50ul, E0840, P/X
 * 3) 5μL, E0840, P/X
 * 4) Blank
 * 5) Blank
 * 6) Blank
 * 7) Blank
 * 8) Blank

Gel purification
the bands were cut with scapel from the gels. the method, book
 * 1) E0840, 900bp
 * 2) R0082, ~3kb

Ligation
To ligate the cognate promnoter R0082 with reporter,E0840.
 * 1) Add 1μL of R0082 backbone
 * 2) Add 7μL of E0840 insert
 * 3) Add 1μL of ligase buffer
 * 4) Add 1μL of ligase
 * 5) Incubate at room temp for 2 hrs

Transformation

 * 1) To 50μL of competent cell (XL-1 Blue)
 * 2) Add 10μL of ligase reaction
 * 3) Place for 5 min on ice
 * 4) Place in the 42°C waterbath for 1 min
 * 5) Put back on ice for 5 min
 * 6) Add 250μL Soc
 * 7) Place in the shaker for 1 hr
 * 8) Plate on agar with appropriate antibiotic (Ampicillin) using glass beads
 * 9) Put in the incubater at 37°C


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