User:Wilfred J. Poppinga/Notebook/Wilfreds Project/2009/07/31

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iGEM promotor oligo's Main project page
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To Do

  • DONE Prepare competent TOP10 Cells
    • DONE Prepare fresh CaCl2 (0.1 M) (0.3319 g in 30 mL demi)
      • DONE Look for filters for sterilization (0.2 μm Whattman filter used to sterilize CaCl2, CaCl2 was put on ice)
    • DONE Look for canister for liquid nitrogen
    • DONE 60 mL (6·10 mL in 50 mL greiner tubes were inoculated with 100 μL ON culture TOP10 cells)
  • DONE Isolate pSB1AC3-H & pSB3K3-H plasmids
    • DONE Nanodrop
  • DONE Transform ligations
    • DONE Use each batch of 20 mL and give 2 batches only positive control

Competent cells

  • 5 mL ON culture is used to inoculate 6·10 mL of LB, 100 μL per 10 mL.
  • Cultures are grown @ 37 °C untill an OD600 of 0.2 ~ 0.3 is reached.
  • Cultures are spinned down @ 4000 rpm, 4 °C
  • Supernatant is removed and pellet is resuspended in 10 mL chilled 0.1 M CaCl2
    • Suspension is incubated on ice for 10 min.
  • Suspensions are spinned down @ 4000 rpm, 4 °C
  • Supernatant is removed and pellet is resuspended in 1770 μL chilled 0.1 M CaCl2 and supplemented with 230 μL 87% glycerol prior to making aliquots.
  • Cells are divided in 50 μL aliquotes
  • Cells are snapfrozen in liquid nitrogen and stored @ -80 °C
OD600
Culture t = 1.25 h t = 1.75 h Cell batch Quality
1
0.119 0.307
2
0.113 0.304
A
Good
3
0.108 0.290
4
0.113 0.301
B
Good
5
0.140 0.315
6
0.120 0.321
C
Good

Plasmid isolation

Plasmids pSB3K3-H and pSB1AC3-H were isolated using (NucleoSpin® Plasmid, Machery nagel]) Concentration was determined using nanodrop

'
Vector ng/μg 260/280
260/230
pSB1AC3-H 116.0 1.80 1.29
pSB3K3-H 23.4 1.36 0.34


Control competence

  • All batches (A, B & CC) were transformed with 1 μL pSB1AC3 isolated today

Oligo's

  • Ligations were transformed in batch B of competent cells, 5 μL ligation mix in 50 μL competent TOP10 cells.
  • 200 μL of LB was added for 1 h of recovery @ 37 °C
  • Suspensions were plated out on TY Amp100 plates
    • 50 μL and 200 μL



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