User:Karmella Haynes/Notebook/BioBrick cloning/2010/01/16

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01/16/10

  • ✓ Minipreps: KAH117, 118/MV2 (re-transformation clones, 2 each)
  • ✓ Cultures: KAH137(3), 138(3)
  • ✓ Assemblies: KAH94, 95/ JDS47; KAH94, 95/ JDS48 (re-do)

Minipreps
> Check with E/P digests

Reagent Volume Expected:
MV2 = 4529
1,2. KAH117/MV2 = 7758
3,4. KAH118/MV2 = 7930
E/P digest 1/16/10
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

--> KAH117/MV2 worked. Pick 4 more colonies from KAH118/MV2 re-transformation plate


Assemblies
--> Re-do (see 1/14), use higher conc. of insert
--> Increase ligation time (1 hr.)

  1. KAH94/JDS47: (1) KAH94/(N, blunted)/5011 + JDS47/(BglII, blunted, dp)/7509 ✓
  2. KAH95/JDS47: (2) KAH94/(N, blunted)/5083 + "
  3. KAH94/JDS48: KAH94/(N, blunted)/5011 + JDS48/(BglII, blunted, dp)/7519 ✓
  4. KAH95/JDS48: KAH94/(N, blunted)/5083 + "

> Digests (Fermentas FD)

Reagent Volume   Cloning digests 1/14/10
30 μL/lane, 1% agarose
DNA (plasmid) 10.0
10x buffer 3.0
NotI 2.0
dH2O 15.0
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s of inserts

Sample OD260 260/280 ng/μL
1. KAH94(N) 0.360 2.57 18.0
2. KAH95(N) 0.363 2.01 18.2


> Blunting (NEB Quick Blunting kit)
--> Blunt all DNA (vectors and inserts)

Reagent Volume
DNA (clean digest) up to 19 μL
10x blunting buffer 2.5
1 mM dNTP mix 2.5
Blunt Enzyme mix 1.0
dH2O ---
  25 μL --> R.T./ 15 min.; 70°C/ 10 min.
  1. KAH94 (max. vol.) [final] = 13.7 ng/μL
  2. KAH95 (max. vol.) [final] = 13.7 ng/μL


> Ligations

Ligation Plate results (lig : neg crtl) 01/17/10
1. KAH94(N, blunted)/5011, 40 ng + JDS47(BglII, blunted, dp)/7509, 30 ng KAH94/JDS47 10:1 (Pick 6)
2. KAH95(N, blunted)/5083, 40 ng + " KAH95/JDS47 10:1 (Pick 6)
3. JDS47(BglII, blunted, dp)/ 30 ng
4. KAH94(N, blunted)/5011, 40 ng + JDS48(BglII, blunted, dp)/7519, 30 ng KAH94/JDS48 10:1 (Pick 6)
5. KAH95(N, blunted)/5083, 40 ng + " KAH95/JDS48 10:1 (Pick 6)
6. JDS48(BglII, blunted, dp)/ 15 ng
  1 2 3 4 5 6
Insert DNA 3.7 3.7 --- 3.7 3.7 ---
Vector DNA 1.3 1.3 1.3 1.3 1.3 1.3
2x lgn buf (Roche) 6.0 6.0 6.0 6.0 6.0 6.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0
dH2O --- --- 3.7 --- --- 3.7
  12μL 12μL 12μL 12μL 12μL 12μL --> R.T./ 1 hr; Add 30 μL Z-DH5α



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