IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week3/Chemical and Light: Difference between revisions
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==Transformations== | ==Transformations== | ||
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. | 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== | ==Ligations== | ||
===mtrA and p45=== | ===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. | 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. | ||
Revision as of 08:13, 8 July 2008
Goals for Week 3
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 |
7/7: 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
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.
