User:Karmella Haynes/Notebook/BioBrick cloning/2010/07/14

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07/14/10

  • ✓ Cultures: pJ10's and pAct5C plasmid transformations (2 mL ~5 hr. Turbo cultures)
  • ✓ Assemblies: re-try cloning from yesterday (may have incorrectly cut V0120)



Minipreps
> Check pJ10's with BamHI/KpnI digests
> Check pAct5C with BioBrick enzymes:

  1. EcoRI/ XbaI
  2. NotI/ XbaI
  3. SpeI/ XbaI
  4. PsI/ XbaI
  5. XbaI
  6. 0 enzyme


Reagent Volume Expected:
1. pJ10:G04380(eGFP) = 760, 4179
2. pJ10:G04376(CFP) = 757, 4179
3. pJ10:G04382(Citrine) = 757, 4179
4. pJ10:G04381(mCh) = 751, 4179
5. pAct5C E/X = ?
6. pAct5C N/X = linear (full length)
7. pAct5C S/X = 2 bands (luciferase, vector)
8. pAct5C P/X = ?
9. pAct5C X = linear (full length)
10. pAct5C ctrl = supercoiled
Miniprep check 7/14/10
15 μL/lane; 1% agarose
DNA(plasmid) 2.0
10X buffer 1.5
enzyme 1 1.0
enzyme 2 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

--> Conclusions: pAct5C contains EcoRI and PstI, but not sure where since there is no sequence data. Will cut out renilla luc w/ SpeI/ NotI and replace with mini BB cloning site (NotI-SpeI). Order primers tomorrow.


Assemblies
> See 07/13/2010

> Digests (Fermentas FD)
--> Re-digest V0120

Reagent Volume  
DNA (plasmid) 25.0
10x buffer 3.0
XbaI 1.0
PstI 1.0
dH2O ---
  30 μL --> 37°C/ ~15 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. V0120 (X/P) 0.642 3.48 32.1


> Ligations

Ligation Plate results (lig : neg crtl) 07/15/10
1. flyNLS(X/P dsOligo)/21, 0.4 ng + V0120(X/P)/3200, 30 ng flyNLS 6 colonies (Pick 2)
2. flyeGFP(X/P PCR)/714, 13 ng + " flyeGFP 13 colonies (Pick 2)
3. flyCFP(X/P PCR)/711, 13 ng + " flyCFP 9 colonies (Pick 2)
4. flyCitrine(X/P PCR)/711, 13 ng + " flyCitrine 6 colony (Pick 2)
2. V0120(X/P)/ 25 ng 1 colony
  1 2 3 4 5
Insert DNA 1.0 1.0 1.0 1.0 ---
Vector DNA 1.0 1.0 1.0 1.0 1.0
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 2.0 2.0 2.0 2.0 3.0
  10 μL 10 μL 10 μL 10 μL 10 μL

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



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