IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week3/Chemical and Light

Summary Powerpoint by Amy

File:7-7monmtg slides (compatible).ppt

Chemical 'n' Light

RE digests

Repeated digests of P1, P18, P26, P38, P39, P45, P56, P57

	P1B	P18	P26A	P38B	P39A	P45B	P56B	P57B
DNA	10 μL	20 μL	3 μL	5 μL	5 μL	3 μL	10 μL	10 μL
10X NEB Buffer	buffer 3, 2.5 μL	buffer 2, 2.5 μL	buffer 3, 2.5 μL	buffer 2, 2.5 μL	buffer 2, 2.5 μL	buffer 3, 2.5 μL	buffer 3, 2.5 μL	buffer 3, 2.5 μL
25X BSA	1 μL	1 μL	1 μL	1 μL	1 μL	1 μL	1 μL	1 μL
REs (1 μL each)	XP	SP	XP	SP	SP	XP	XP	XP
Water	9.5 μL	0 μL	16.5 μL	14.5 μL	14.5 μL	16.5 μL	9.5 μL	9.5 μL

07/07: More CDF was PCRed: 45μL PCR supermix, 1μL S1P13 (435ng/μL), 1μL CDF-F primer (20μM), 1μL CDF-R primer (20μM), 2μL water. Conditions: 5min @ 94°C → 35x[45s @ 94°C → 45s @ 58.7°C → 1m45s @72°C] → 5min @ 72°C → ∞ @ 4°C, heated lid

07/08: Digested pCDF with XP:

• 12.5 μL water
• 1 μL 25X BSA
• 2.5 μL 10X NEB Buffer 3
• 7 μL DNA
• 1 μL of each RE (XbaI and PstI)

07/08: All of these digested plasmids were run on a 1% low melt gel and were extracted using the following protocol:

• Cut out the bands
• Freeze at –20C for at least 20'
• Spin 13000 RPM 10’
• Use supernatant for ligation
• Then do gel extraction on rest of gel (QIAGEN protocol) and combine with above

07/08: We ligated the following fragments and transformed them into DH5α cells (the vector is listed before the insert:

• P38 and P56
• P38 and P57
• P39 and P56
• P39 and P57
• P1 and P26
• P18 and P45
• P1 and pCDF

The ligation protocol is

• 2 μL 5X Dilution Buffer
• 2 μL vector
• 6 μL insert
• 10 μL 2X Rapid Ligation Buffer
• 1 μL ligase
• vortex briefly
• incubate at RT for 10'
• use 5 μL to transform 50 μL cells

Transformations

07/07: We transformed P42, P11, P51, P52, P77, P78, P17, Q01121, BBa_J06911, BBa_J06912 from both the 2007 and 2008 registries (P11 was available only from the 2008 registry). We also made a positive transformation control using pUC19 DNA that came with the TOP10 cells. We made three negative plate controls using mock transformed bacteria on Amp, Kan, and Cm plates. We used E1.

Ligations

mtrA and p45

Ligated mtrA and p45 using standard protocol from ligation kit. In step 2 5uL of Vector DNA was added to make a 20uL dephosphorylation mix. In step 6, 4uL of the dephosphorylated Vector DNA and 4uL insert DNA were added.