Haynes Lab:Notebook/Engineering PC-TFs/2014/07/07

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10ever Lab

Restriction Digest and Gel Extraction
Gel extracted BL01, BL05, BL09 from V0120 and re-verified colony 1 BL01, BL05, BL09.

Reagent Volume
DNA(BL01, BL05, BL09 respectively) 20.0 μL
10X buffer 3.0 μL
XbaI 1.0 μL
SpeI 1.0 μL
dH2O 5.0 μL
Total 30 μL --> 37°C/ 30 min.




Gel Verification
On the same gel.

Reagent Volume
DNA(BL01, BL05, BL09 respectively) 5.0 μL
10X buffer 1.5 μL
PstI 1.0 μL
EcorI 1.0 μL
dH2O 6.5 μL
Total 15 μL --> 37°C/ 30 min.


Gel exploded so I did not take a picture of it. I had mislabeled BL09 as BL05.


Gel Purification

Cut expected bands from gel. Assumed 200mg which lead to using 600μL ADB during standard gel purification procedure.

Plasmid OD260 OD260/280 ng/μL
1. BL01 C2 0.008 1.55 7.94
1. BL05 C2 0.007 1.797 7.425
1. BL09 C2 0.007 1.5 7.47




Ligation Calculations resulted in the following:

  Ligation Negative Control
Insert DNA (BL01, BL05, BL09 respectively) 12μL none
Vector DNA (50 ng) 1μL same
10x Biolabs T4 Ligase Buffer 2.0 μl same
Biolabs T4 Ligase 1.0 μl same
dH2O 4.0μL 16.0μL
  20.0 μL total    same


Mix the reaction(s) thoroughly by flicking the tube.
Incubate at room temperature for 30 minutes.


Long Transformation
Using the set up ligation products from above.

  • DH5α-T cells were thawed in ice.
  • Six 1.5mL tubes were setup and labeled; 1 (BL01), 5 (BL05), 9 (BL09), CMV9 (CMV/MV9, ligation benchmark), rhlI (pos control), Neg (negative control).
  • 50μL of thawed cells were transferred into each of these six empty tubes and pipetted up and down to mix.
  • The ligation products were then transferred into their respective tubes and flicked to mix. Set back on ice.
  • A 1:1000 RhlI plasmid was used as a positive control; 1μL of this plasmid was added to 19μL dH2O to equal the other two products' 20μL total volume. Added to the fifth tube with 50μL thawed cells.
  • All tubes incubated on ice for 30 minutes.
  • All three tubes were heat shocked on the heat block at 42°C for 30 seconds and then placed back on ice for 2 minutes.
  • 900μL LB broth (no antibiotic) was pipetted into each of the three tubes.
  • Tubes were taped into an empty plate. The plate was taped to the shaker within the incubator.
  • Incubated on shaker for 1 hour at 37°C and 240rpm.


Did NOT:

  • Tubes centrifuged for 2 minutes at 6000 x g.
  • 600μL supernatant discarded from each tube.
  • Vortexed tubes to re-suspend cells.


DID:

  • 300μL of re-suspended cells were then plates on six marked plates and placed in the incubator for overnight growth.



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