IGEM:MIT/2007/Notebook/2007-7-17

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(Digestion of (F2620 and B0034) for 3A)
(Digestion of (F2620 and B0034) for 3A)
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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 12:08, 17 July 2007

Contents

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
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