User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/02

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12/02/09

  • ✓ Minipreps: KAH011(8); sequencing order
  • ✓ Digests: check MV2
  • ✓ Assembly: Re-do KAH93, 94, 95 in MV2 (longer MV2 digest, dephos)

Minipreps
> Check via sequencing (Genewiz)


Digests
> Cloning with MV2 gives high background
> Check MV2 w/ EcoRI, PstI and SpeI individually

Reagent Volume Expected = 4529
Lanes:
1. uncut (0 enzyme)
2. EcoRI
3. PstI
4. SpeI
File:KAH 120209 gel1.tif
15 μL/lane, 1% agarose
DNA (plasmid) 1.0
10x buffer 1.5
enzyme 1.0 (0 for control)
dH2O 11.5
  15 μL --> 37°C/ ~30 min.

Note: Looks like all three cut fine. For cloning (E/P), try a longer digest with more enzyme. Also dephos. the cut vector.


Assemblies

  1. KAH93/MV2: (1) KAH93/(E/P)/4839 + (4) pcDNA3.1+puro/ΔCMV(MV2)/(E/P)/4529
  2. KAH94/MV2: (2) KAH94/(E/P)/5011 + "
  3. KAH95/MV2: (3) KAH95/(E/P)/5083 + "

> Digests (Fermentas FD)

Reagent Volume  
DNA (plasmid) 10.0
10x buffer 3.0
EcoRI 2.0
PstI 2.0
dH2O 13.0
  30 μL --> 37°C/ ~1 hr.; Zymo clean


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. MV2(E/P) 1.441 1.85 72.0


> Dephosphorylation (Roche) --> Do 2 rxn's

Reagent Volume
DNA (clean digest) 7.0 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O 10.0
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


> Ligations

Ligation Plate results (lig : neg crtl) 12/03/09
1. KAH93(E/P)/4839, 43 ng + MV2(E/P)/4529, 20 ng KAH93/MV2 10:1 (Pick 2)
2. KAH94(E/P)/5011, 44 ng + " KAH94/MV2 10:1 (Pick 2)
3. KAH95(E/P)/5083, 45 ng + " KAH95/MV2 10:1 (Pick 2)
4. MV2(E/P)/ 20 ng  
  1 2 3 4
Insert DNA 1.3 2.3 4.2 ---
Vector DNA 0.8 0.8 0.8 0.8
2x lgn buf (Roche) 6.0 6.0 6.0 6.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0
dH2O 2.9 1.9 --- 4.2
  12μL 12μL 12μL 12μL --> R.T./ ~10 min.; Add 25μL Z-DH5α