User:Wilfred J. Poppinga/Notebook/Wilfreds Project/2009/08/19

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 * style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]] Project name
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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GVP

 * ✅ Make picture of floating bacteria

Metal promotors

 * Look into follow up

Protocol #3
Restriction vectors
 * Mix
 * 2 μL Tango digest buffer (Fermentas)
 * 16 μL vector
 * 1 μL EcoRI (Non-fast digest)
 * 1 μL SpeI (Non-fast digest)
 * Incubate 1.5 h @ 37 °C
 * Put to 1% agarose (1xTBE)
 * Isolate ~3000 bp vector
 * Check for presence of cut out ccdB death gene (~600 bp)
 * Purify vector using gel purification kit
 * End volume 30 μL H2O (MilliQ)
 * Determine concentration using nanodrop
 * Store to 4 °C until ligation

Phosphorylation of 5' ends & hybridization [1] 
 * (SENSE) Mix:
 * 3 μL 100 µM sense oligo
 * 1 μL 10 x PNK (polynucleotide kinase) buffer (Fermentas Buffer A)
 * 2 μL 10mM ATP
 * 1 μL T4 polynucleotide kinase (PNK)
 * 3 μL MilliQ
 * (for selfcloser control, do not add oligo's. Instead 6 μL MilliQ in total)
 * (ANTI-SENSE) Mix:
 * 3 μL 100 µM anti-sense oligo
 * 1 μL 10 x PNK (polynucleotide kinase) buffer (Fermentas Buffer A)
 * 2 μL 10mM ATP
 * 1 μL T4 polynucleotide kinase (PNK)
 * 3 μL MilliQ
 * (for selfcloser control, do not add oligo's. Instead 6 μL MilliQ in total)
 * Incubate @ 37 °C for 1.5 hours.
 * Mix
 * 10 μL Sense mixture
 * 10 μL Anti-sense mixture
 * 3 μL 0.5 M NaCl
 * Place in boiling water for 3 min., and allow the reaction to cool to room temperature.

Ligation
 * Mix
 * 1 μL T4 ligase buffer
 * 7.5 μL restricted vector (purified from gel)
 * 1 μL annealing mix
 * 0.5 μL T4 ligase
 * Incubate
 * 1h RT
 * or
 * ON @ 4 °C

Transformation
 * Add 5 μL of ligation mixture to 50 μL of TOP10 chemically competent cells
 * Heatshock, 45 sec. 42 °C
 * + control: 1 μL pSB3K3-high or pSB1AC3-high plasmid, - control: 1 μL MilliQ
 * Alternatively a single cut plasmid can be taken as a ligation control
 * Alternative - control: 1 μL pSB1AC3 or pSB3K3 carrying ccdB deathgene
 * Incubate 1 h @ 37 °C, 200 RPM
 * Plate out on LB-agar + Kanamycin (30 μg/ml for pSB3K3) or Ampicillin (100 μg/mL for pSB1AC3)
 * Plate out 50 μL & 200 μL of cell suspension
 * Grow ON @ 37 °C

Checking transformations
 * See if - control is empty for functioning antibiotics and death gene
 * See how many colonies on + control for functioning competent cells
 * See how many selfclosers and compare to samples (>10x on sample vs. selfcloser)
 * If enough transformants, inoculate 3 - 5 colonies in an ON culture
 * Alternatively perform colony PCR

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{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Project name
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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

FAKIN

 * Mail Proost

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