User:Karmella Haynes/Notebook/BioBrick cloning/2010/07/27

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07/27/10

  • ✓ Assembly: start re-cloning pAct5C/microMCS (from 7/25/10)
  • ✓ Cultures/ minipreps: KAH015 (4, 2 mL)



Digests
> Mut mCh clones from last week
> Check with X/P digests (unique XbaI introduced by microMCS insert)
> Will need to confirm proper orientation of microMCS by sequencing

Reagent Volume Expected:
1-4.KAH015 = 649, 7753
Digest check 7/27/10
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
BamHI 1.0
KpnI 1.0
PstI 1.0
dH2O 8.5
  15 μL --> 37°C/ ~15 min.

--> Send all 4 to Genewiz for sequencing on Wednesday


Assembly
--> re-do pAct5C/microMCS (from 7/25/10)

  1. KFV1: (1) pAct5C/(S/N)/~7000 + microMCS/(ΔN/ΔS dsOligo)/~35


> Dephosphorylation (Roche)
--> pAct5C/(S/N)

Reagent Volume
DNA (clean digest) 15.0 (120 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O 2.0
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 6 ng/μL


>Kinase rxn. and Oligo annealing
--> Need to phosphorylate microMCS before inserting into dp vector (overnight)

Reagent Volume
100 μM oligos (3.0 μL each, up to 21 μL)
10x PNK buffer (NEB) 3.0
10 mM ATP 2.0
T4 PNK 2.0
dH2O 2.0
  30 μL --> 37°C/ 1 hr.; Add 4.0 μL 500 mM NaCl; 100 °C 10 min.; cool to R.T. o/n


7/28/10
> Ligations

Ligation Plate results (lig : neg crtl) 07/27/10
1. microMCS(ΔS/ΔN dsOligo)/35, 0.25 ng + pAct5C(S/N)/7000, 25 ng KFV1 2 colonies
2. pAct5C(S/N), 25 ng 0 colonies
  1 2
Insert DNA 0.5 ---
Vector DNA 4.5 4.5
2x lgn buf (Roche) 6.0 6.0
T4 ligase (NEB) 1.0 1.0
dH2O --- 0.5
  12μL 12μL



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