User:Karmella Haynes/Notebook/BioBrick cloning/2010/02/02

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02/02/10

  • ✓ Colony PCR: from EF-1α site directed mutagenesis plates, PCR w/ BioBrick primers
  • ✓ Assembly: EF-1α into V0120



Colony PCR & BioBricking
> Pick colonies from site directed mutagenesis plates from 2/01 (6 each)
> Use Forward EF1a/ Reverse EF1a (contain Xba and Pst ends)
> Cut products with X/P (PstI leaves desired mutants in tact and cuts non-mutated fragments)
> Isolate 1184 bp band for cloning


PCR
1-6. Forward strand mutagenesis clones
7-12. Reverse strand mutagenesis clones
13. EF-1α (original template plasmid DNA)

Reagent Volume
Colony pick 0.0
10 μM primer mix 1.0
2x GoTaq Green 12.5
dH2O 11.5
  25 μL

--> BioRad 96-well

  • 95°C/ 10 min.
  • [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 1 min.] x30
  • 72°C/ 3 min.
  • 4°C/ ∞

--> Zymo clean & concentrate; elute w/ 20 μL dH2O


Digest
--> Digest samples w/ XbaI/PstI

Reagent Volume Expected:
1-6. Fwd strand mut clones = 1184
7-12. Rev strand mut. clones = 1184
13. EF-1α PCR (uncut)
X/P PCR digest 2/02/10
25 μL/ lane; (#13, 10 μL); 1% agarose
DNA(clean PCR) 20.0
10x buffer 2.5
XbaI 1.0
PstI 1.0
dH2O 0.5
  25 μL --> 37°C/ 30 min.

--> Gel purify for Assemblies



Assemblies

  1. EF-1α(1): ✓ EF-1α PCR 1/(X/P)/1184 + ✓ V0120/(X/P)/3200
  2. EF-1α(2): ✓ EF-1α PCR 2/(X/P)/1184 + "
  3. EF-1α(3): ✓ EF-1α PCR 3/(X/P)/1184 + "

> Measure conc.'s

Sample OD260 260/280 ng/μL
1. EF-1α PCR 1(X/P) 0.191 1.81 9.5
2. EF-1α PCR 2(X/P) 0.143 1.74 7.2
3. EF-1α PCR 3(X/P) 0.229 1.70 11.4


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. EF-1α PCR 1(X/P)/1184, 11 ng + V0120(X/P)/3200, 15 ng EF-1α-1 0 colonies
2. EF-1α PCR 2(X/P)/1184, 11 ng + " EF-1α-2 1 colony (Pick 1)
3. EF-1α PCR 3(X/P)/1184, 11 ng + " EF-1α-3 5 colonies (Pick 3)
2. V0120(X/P)/ 15 ng 0 colonies
  1 2 3 4
Insert DNA 2.5 2.5 2.5 ---
Vector DNA 1.5 1.5 1.5 1.5
2x lgn buf (Roche) 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0
dH2O --- --- --- 2.5
  10μL 10μL 10μL 10μL --> R.T./ ~10 min.; Add 25 μL z-DH5α



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