User:Karmella Haynes/Notebook/BioBrick cloning/2010/07/25

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07/25/10

  • ✓ Assemblies: microMCS sites into FUW-Oct4 and pAct5C
  • ✓ Miniprep digests: check mut. mCh clones from last week



Digests
> Mut mCh clones from last week
> Check with BamHI/KpnI/PstI digests (plasmid = pJ10:G04381)

Reagent Volume Expected:
mut = 751, 3639
wt = 388, 363, 3639
File:KAH 072510 gel1.tif
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 Monday


Assemblies

  1. KFV1: (1) pAct5C/(S/N)/~7000 + microMCS/(ΔN/ΔS dsOligo)/~35
  2. KAH015: (2) TetO-FUW-Oct4/(E)/8380 + microMCS2/(E/ΔE dsOligo)/~20


>Kinase rxn. and Oligo annealing
--> Forgot to order microMCS2 oligos with 5' phosphates; re-do annealing rxn.

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


> Digests (Fermentas FD)
--> FUW-Oct4 EcoRI fragment = 1316
--> pAct5C SpeI/NotI fragment (R. luciferase) = ~1000

Reagent Volume   File:KAH 072510 gel2.tif
30 μL/lane, 1% agarose
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. pAct5C(S/N) 0.160 1.38 8.0
2. TetO-FUW-Oct4(E) 1.569 1.78 78.5


> Dephosphorylation (Roche)
--> TetO-FUW-Oct4/(E)

Reagent Volume
DNA (clean digest) 6.4 (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O 10.6
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


> 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 1:1
2. pAct5C(S/N), 25 ng ~30 colonies
3. microMCS2(E/ΔE dsOligo)/25, 0.15 ng + TetO-FUW-Oct4(E)/8380, 25 ng KAH015 5:1 (Pick 4)
4. TetO-FUW-Oct4(E), 25 ng ~5 colonies
  1 2 3 4
Insert DNA 0.5 --- 0.5 ---
Vector DNA 3.5 3.5 1.0 1.0
2x lgn buf (Roche) 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0
dH2O --- 0.5 2.5 3.0
  10μL 10μL 10μL 10μL

--> Re-do pAct5C/microMCS cloning with dp vector