Haynes Lab:Notebook/Engineering PC-TFs/2014/05/15: Difference between revisions

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Line 26: Line 26:
| bgcolor=#cfcfcf | Reagent  
| bgcolor=#cfcfcf | Reagent  
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <u>Expected:</u><br>1. CMV = 2500bp <br>
| rowspan="7" | <u>Expected:</u><br>1. BL01 = 2500bp <br>
|-
|-
| DNA(BL01) || 15.0 μL
| DNA(BL01) || 15.0 μL

Revision as of 20:39, 15 May 2014



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Summary

Digest PcTF1 (BL01)


Restriction Digest Table

Reagent Volume Expected:
1. BL01 = 2500bp
DNA(BL01) 15.0 μL
10X buffer 3.0 μL
XbaI 1.0 μL
SpeI 1.0 μL
dH2O 10 μL
Total 30 μL --> 37°C/ 15 min.


Gel Purification: CMV/V0120

  • Using Prior Digest
  • Placed 15μL 1kB Ladder in well one and 30μL of BL01 digest in well two.
  • Ran gel at 110V for 55 minutes.
  • Placed gel on UV transilluminator and cut out desired band.
Hover name
0.8% agarose; cut the brightest band furthest down at ~ 2500bp; Ladder
  • Used Zymo gel purification kit.
  • Assumed 200mg gel, added 600μL ADB to the 1.5mL tube.
  • Put on heat block at 55°C for 5 minutes, vortexed and then placed back on the heat block for an additional 5 minutes, vortexed again.
  • Put ~750μL gel/ADB into spin column, centrifuged at max rpm for 30 seconds.
  • Pipetted 200μL wash buffer to the tube, centrifuged at max rpm for 30 seconds. Repeated once.
  • Transferred column to new labeled 1.5mL tube.
  • Pipetted 15μL elution buffer to the column.
  • Spun at max rpm for 30 seconds.
  • Put marked (GelP BL01 5/15/2014) BL01 in -20°C fridge in box labeled Cameron.

Ligation

  • Added 10μL dH2O to two 0.5mL tubes marked 1 and 2.
  • Pipetted 0.5μL BL01 into tube 1. Put both tubes on ice.
  • Let DH5a-T cells thaw in ice.
  • Transferred 50μL cells into each tube. Incubated for 5 minutes on ice.
  • Spread sample 1 onto labeled plate 1 using sterile beads. Repeated for tube two and plate two.
  • Placed in incubator for overnight growth.


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