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

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

Plasmid isolation

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

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