User:Wilfred J. Poppinga/Notebook/Wilfreds Project/2009/07/28

{| width="800"
 * style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]] Metal dependent promotors
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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

Restriction vectors
Vectors: pSB3K3 & pSB1AC3

Incubate 0.5 h @ 37 °C  Bring vectors to 1% agarose gel and cut out with scalpel, purify using gel purification kit (NucleoSpin® Extract II, Machery nagel) to an end volume of 50 μL. (alternatively, Phosphatase treatment of linearized vector)
 * 10 μL 10x Fast digest buffer (Fermentas)
 * 2 μL SpeI (Fast digest, Fermentas)
 * 2 μL EcoRI (Fast digest, Fermentas)
 * 50 μL DNA (pSB3K3 or pSB1AC3)
 * 36 μL MilliQ

Phosphorylation of 5' ends & hybridization [1]

 * Mix:
 * 3 μL 100 µM sense oligo
 * 3 μL 100 µM anti-sense oligo
 * 3 μL 10 x PNK (polynucleotide kinase) buffer (Fermentas Buffer A)
 * 2 μL 10mM ATP
 * 2 μL T4 polynucleotide kinase (PNK)
 * 17 μL MilliQ
 * (for selfcloser control, do not add oligo's. Instead 23 μL MilliQ in total)

to give 30 μL total volume 
 * Incubate @ 37 °C for 1.5 hours.
 * Add 4 μL 0.5 M NaCl.
 * Place in thermocycler (99 °C) for 3 min., and allow the reaction to cool to room temperature.

Ligation

 * Mix the vector (3 μL) and the annealing mix 1 μL and then add ligase buffer (1 μL) and ligase (1 μL) (and 4 μL MilliQ) once the mix gets close to room temperature. this should reduce the likelihood of insert multimers forming [2]</SUP>.
 * As the paired oligos cool, they will also form multimers of your insert. To release them, you should heat the mixture of your vector and insert DNA to about 65 °C and let it cool prior to adding ligase<FONT SIZE="-1"> [2]</FONT></SUP>.

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
 * 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
 * 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

Plasmid isolation and quality control

 * Isolate plasmids and perform restriction analysis

Protocols

 * 1) Koch_Lab:Protocols/Oligonucleotide Annealing/Duplexes
 * 2) Silver: Oligonucleotide Inserts
 * 3) Annealing complementary primers
 * 4) Endy:Annealing complementary primers
 * 5) PNK Treatment of DNA Ends
 * 6) DNA ligation

Results
July 28th 2009


 * }