IGEM:MIT/2007/Notebook/2007-7-17: Difference between revisions
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**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 | **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 | ||
===Miniprep Results for 5mL LCs of | ===Miniprep Results for 5mL LCs of pT040 made on 7/16=== | ||
<pre> | <pre> | ||
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4 57.9 ng/µL | 4 57.9 ng/µL | ||
</pre> | </pre> | ||
eppendorfs containing the purified plasmids are labeled: | |||
iGEM 7/17 | |||
<br> | |||
pur. pT040 | |||
<br> | |||
LC # | |||
<br> | |||
Concentration |
Revision as of 10:40, 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
37µl (F2620 and B0034) @ 27ng/µl 0.5µl BSA 5µl NEB2 Buffer 1µl EcoRI 1µl SpeI 5.5µl H2O ------------------ 50µl Total
- Digested pSB1AC3: 15µl @ 125 ng/µl
- Digestions placed in 37C @ 11am
Changes to the OWW "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
Miniprep Results for 5mL LCs of pT040 made on 7/16
Colony DNA Conc. 1 60.6 ng/µL 2 64.3 ng/µL 3 78.2 ng/µL 4 57.9 ng/µL
eppendorfs containing the purified plasmids are labeled:
iGEM 7/17
pur. pT040
LC #
Concentration