Restrictions

Restricted (total 60µL DNA) K314100 in pSB1C3 with Spe1 and Pst1
Restricted (total 60µL DNA) Oxytocin in puc19a with Xba1 and Pst1
Restricted (total 60µL DNA) NEC1puc19a with Xba1 and Pst1
Set 12 µL aside from each reaction and gel extracted (GEX) the remaining 48µL
Combined heat killed restriction mixtures into ligation mix from NEB using NEB protocol, left at room temperature for 10 mins then heat killed for 20 mins at 80°C
K314100 – pSB1C3 (s,p), with: Oxytocin (x,p)
K314100 – pSB1C3 (s,p), with: NEC1 (x,p)
GEX K314100 – pSB1C3 (s,p), with: GEX Oxytocin (x,p)
GEX K314100 – pSB1C3 (s,p), with: GEX NEC1 (x,p)

Transformation

Transformed ligations into 20µL high efficiency DH5α cells and 50µL lab made competent DH5α
- K314100 – pSB1C3 (s,p), with: Oxytocin (x,p)
- K314100 – pSB1C3 (s,p), with: NEC1 (x,p)
- GEX K314100 – pSB1C3 (s,p), with: GEX Oxytocin (x,p)
- GEX K314100 – pSB1C3 (s,p), with: GEX NEC1 (x,p)
Plated on CAM plates
Results: Only the 20µL non gel extracted DH5α E. coli high efficiency cells grew

Date: June 4, 2013
Picked 10 colonies from each plate
Grew over night in LB and mini prepped
PCR-ed with VF2 and VR primers, no results as shown
Picked 6 Oxytocin colonies and 5 NEC1 colonies grew overnight in LB – mini prepped and restricted all colonies picked so far at E, P cute sites and ran on gel, results concluded as ligations did not work since a band is seen at ~500 bp size of the promoter construct K314100 but not with oxytocin or NEC1

Date: June 10, 2013
Sent 10µL DNA with 5µL of 5µM VF2 primer to genewize
- J23100-J06702
- J23108-J06702
- J23113-J06702
- K314100-oxytocin neurophysin1
June 12th results arrived
- J23100-J06702- positive for J06702 but no promoter found
- J23108-J06702- positive for J06702 but no promoter found
- J23113-J06702- positive for J06702 but no promoter found
- K314100-oxytocin neurophysin1 – Positive results full sequence found

Date: June 13, 2013
Transform mini prepped plasmid DNA of K314100–Oxytocin Neurophysin1 in pSB1C3 into 15µL of Bl21 E.coli cells
Let grow over night at 37°C on CAM plates

Date: June 13, 2013
Restricted K314100 Spe1 and Pst1 and NEC1 at Xba1 and Pst1 for 1 hour at 37°C followed by 20 min heat kill at 80°C
Mixed restrictions in ratio of 10µL NEC1 and 2µL k314100 in pSB1C3 let sit in ligation mixture for 10 mins at room temperature followed by 20 min heat kill at 80°C
Transformed 2µL into 20µL of DH5α E.coli highly competent cells, incubated overnight at 37°C

Date: June 13, 2013
K314100–Oxytocin neurophysin 1 in pSB1C3 grew in Bl21 from last nights transformation, picked 4 colonies and grew up in 5ml of LB overnight at 37°C with shaking