IGEM:MIT/2007/Notebook/2007-7-17: Difference between revisions
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*Digested pSB1AC3: 15µl @ 125 ng/µl | *Digested pSB1AC3: 15µl @ 125 ng/µl | ||
*Digestions placed in 37C @ 11am | *Digestions placed in 37C @ 11am | ||
===Changes to the "Transforming Chemically Competent Cells" Protocol for TK Cells=== | |||
*All steps are the same, except for the following: | |||
**In step 2, Note -- TK said that less than 5% of the solution should be cell volume | |||
**Step 4 -- change to 45 to 50 seconds at 42 degrees | |||
**Step 7 -- The 45 incubation time is okay for Kan and Amp resistances, but should be increased to 1.5 to 2 hours for Tet or Cu resistant cells |
Revision as of 09:08, 17 July 2007
Agenda
- Check sequencing results of second (uncontaminated) set of miniprepped 3K3+F2620+B0034
- Digest CPX, 3K3+F2620+B0034 (if sequencing is correct), and a hi-copy plasmid for 3A assembly
- Ligate digested CPX, 3K3+F2620+B0034, and hi-copy plasmid
- Test Standard Assembly of pSB3k3 with F2620, B0034, E1010 using AHL
- LC grown overnight
Digestion of CPX for 3A
5µl CPX (diluted to 1µg/5µl) 0.5µl BSA 5µl NEB3 Buffer 1µl XbaI 1µl PstI 37.5µl H2O ------------------ 50µl Total
Digestion of (F2620 and B0034) for 3A
20µl (F2620 and B0034) @ 35ng/µl 0.5µl BSA 5µl NEB2 Buffer 1µl EcoRI 1µl SpeI 22.5µl H2O ------------------ 50µl Total
- Digested pSB1AC3: 15µl @ 125 ng/µl
- Digestions placed in 37C @ 11am
Changes to the "Transforming Chemically Competent Cells" Protocol for TK Cells
- All steps are the same, except for the following:
- In step 2, Note -- TK said that less than 5% of the solution should be cell volume
- Step 4 -- change to 45 to 50 seconds at 42 degrees
- Step 7 -- The 45 incubation time is okay for Kan and Amp resistances, but should be increased to 1.5 to 2 hours for Tet or Cu resistant cells