User:Karmella Haynes/Notebook/BioBrick cloning/2010/07/13

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07/13/10

  • ✓ PCR: flyeGFP, flyCitrine, flyCFP
  • ✓ Assemblies: dsOligo flyNLS, PCR flyeGFP, flymCh, flyCitrine, flyCFP into V0120
  • ✓ Transformation: transform DH5α Turbo with plasmids for



PCR

  1. flyeGFP (714 bp)
  2. flyCFP (711 bp)
  3. flyCitrine (711 bp)
Reagent Volume
DNA(plasmid) 0.4
10μM primer mix 1.0
2x GoTaq 25.0
dH2O 23.6
  50.0 μL

BioRad PCR Block A

  • 95°C/ 3 min.
  • [95°C/ 30 sec.; 57°C/ 10 min.; 72°C/ 10 min.] x 30
  • 72°C/ 3 min.
  • 4°C/ ∞

> Zymo clean & concentrate
--> Elute w/ 25 μL dH2O
> Proceed to digestion



Assemblies

  1. KHF01/V0120: ✓ flyNLS/(X/P dsOligo)/21 + (1) V0120/(X/P)/~3200
  2. KHF02/V0120: (2) flyeGFP/(X/P PCR)/714 + "
  3. KHF03/V0120: (3) flyCFP/(X/P PCR)/711 + "
  4. KHF04/V0120: (4) flyCitrine/(X/P PCR)/711 + "


> Digests (Fermentas FD)

Reagent Volume   Cloning digest 7/13/10
30 μL/lane, 1% agarose
DNA (plasmid) up to 25 μL
10x buffer 3.0
XbaI 1.0
PstI 1.0
dH2O ---
  30 μL --> 37°C/ ~10 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. V0120 (X/P) 0.271 2.13 13.5
2. flyeGFP PCR (X/P) 1.060 1.86 53.0
2. flyCFP PCR (X/P) 0.641 1.71 32.0
2. flyCitrine PCR (X/P) 0.960 1.78 48.0


> Ligations

Ligation Plate results (lig : neg crtl) 07/14/10
1. flyNLS(X/P dsOligo)/21, 0.3 ng + V0120(X/P)/3200, 25 ng KHF01 2 colonies
2. flyeGFP(X/P PCR)/714, 11 ng + " KHF02 colonies
3. flyCFP(X/P PCR)/711, 11 ng + " KHF03 5 colonies
4. flyCitrine(X/P PCR)/711, 11 ng + " KHF04 1 colony
2. V0120(X/P)/ 25 ng 0 colonies
  1 2 3 4 5
Insert DNA 1.0 1.0 1.0 1.0 ---
Vector DNA 2.0 2.0 2.0 2.0 2.0
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0
dH2O 1.0 1.0 1.0 1.0 2.0
  10 μL 10 μL 10 μL 10 μL 10 μL

--> R.T./ 10 min.; Add 40 μL DH5α Turbo

--> Results: colonies suspiciously low. Not sure if I mistook EcoRI for XbaI tube when cutting V0120. Do over.


Plasmid Transformation
> Plasmids from the DePace lab

  1. pJ10:G04380 (flyeGFP+NLS)
  2. pJ10:G04376 (flyCFP+NLS)
  3. pJ10:G04382 (flyCitrine+NLS)
  4. pJ10:G04381 (flymCherry+NLS)
  5. pAct5C
  6. dH2O (neg ctrl)

> Transform 0.5 μL (in 10 μL dH2O) into 30 μL DH5α Turbo; plat on 100 μg/mL Amp



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