User:Wilfred J. Poppinga/Notebook/Wilfreds Project/2009/07/30

{| width="800"
 * 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


 * colspan="2"|
 * colspan="2"|

Checking transformants after ON incubation
Probably the ligase (buffer) has gone bad, new was fetched and buffer was aliquoted to minimize thawing
 * Positive controls full
 * Negative controls (including self closers) empty
 * Everything else empty
 * After letting plates in incubator the whole day some colonies seemed to appear, probably not much good?

Restriction/Ligation

 * New vector (pSB1AC3 and pSB3K3) was restricted using SpeI and EcoRI
 * pSB3K3 disappeared on gel, due to the high dilution with loading buffer
 * pSB1AC3 was purified (18.5 ng/μL)
 * Due to limited amount of competent cells ligation was set O.N. @ 4 °C
 * protocol was repeated, using oligo's phosphorylated and annealed before ligation was put in fridge

Miscellaneous

 * Plated out MBP transformants
 * 100 μL on LB+Amp (120 μg/mL) plate
 * and quick spin, resuspending pellet in 100 100 μL and putting 100 μL on LB+Amp (120 μg/mL) plate
 * New pSB1AC3-H and pSB3K3-H plasmids were put to culture (2x 3 mL) from positive control plates


 * }