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

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 * style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]] iGEM promotor oligo's
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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To Do

 * ✅ Prepare competent TOP10 Cells
 * ✅ Prepare fresh CaCl2 (0.1 M) (0.3319 g in 30 mL demi)
 * ✅ Look for filters for sterilization (0.2 μm Whattman filter used to sterilize CaCl2, CaCl2 was put on ice)
 * ✅ Look for canister for liquid nitrogen
 * ✅ 60 mL (6·10 mL in 50 mL greiner tubes were inoculated with 100 μL ON culture TOP10 cells)
 * ✅ Isolate pSB1AC3-H & pSB3K3-H plasmids
 * ✅ Nanodrop
 * ✅ Transform ligations
 * ✅ 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


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