User:Karmella Haynes/Notebook/BioBrick cloning/2010/01/14

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01/14/10

  • ✓ Minipreps: KAH117, 118/MV2 (6 each); KAH94 (2), KAH95 (2) retransformations
  • ✓ Assemblies: KAH94, 95/JDS47; KAH94, 95/JDS48
  • ✓ Re-transform: KAH117, 118/MV2

Minipreps
> Check with E/P digests

Reagent 1-12 13-16 Expected:
1-6. KAH117/MV2 = 7758
7-12. KAH118/MV2 = 7930
13,14. KAH94 = 5011
15,16. KAH95 = 5083
File:KAH 011410 gel1.tif
15 μL/lane; 1% agarose
DNA(plasmid) 5.0 2.0
10X buffer 1.5 1.5
EcoRI 1.0 1.0
PstI 1.0 1.0
dH2O 6.5 9.5
  15 μL 15 μL --> 37°C/ ~15 min.

--> Re-transform 117, 118/MV2 to isolate clones without mysterious extra bands


Assemblies

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

> Digests (Fermentas FD)

Reagent Volume   File:KAH 011410 gel2.tif
30 μL/lane, 1% agarose
DNA (plasmid) 5.0
10x buffer 3.0
NotI 2.0
dH2O 20.0
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s of inserts

Sample OD260 260/280 ng/μL
1. KAH94(N) 0.085 1.80 4.3
2. KAH95(N) 0.073 2.08 3.7


> 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] = 3.3 ng/μL
  2. KAH95 (max. vol.) [final] = 2.8 ng/μL
  3. JDS47 (use max. vol, measure after cleaning)
  4. JDS48 (use max. vol, measure after cleaning)


> Clean vector DNA using Zymo DNA Clean & Concentrator

Sample OD260 260/280 ng/μL
3. JDS47(BglII) 0.286 1.79 25.6
4. JDS48(BglII) 0.817 1.85 40.9


> Dephosphorylation (Roche)

Reagent Volume
DNA (clean) up to 17 μL (400 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 20 ng/μL


> Ligations

Ligation Plate results (lig : neg crtl) 01/15/10
1. KAH94(N, blunted)/5011, 20 ng + JDS47(BglII, blunted, dp)/7509, 15 ng KAH94/JDS47 0 colonies (re-do)
2. KAH95(N, blunted)/5083, 20 ng + " KAH95/JDS47 0 colonies (re-do)
3. JDS47(BglII, blunted, dp)/ 15 ng 3 colonies
4. KAH94(N, blunted)/5011, 20 ng + JDS48(BglII, blunted, dp)/7519, 15 ng KAH94/JDS48 1 colony (re-do)
5. KAH95(N, blunted)/5083, 20 ng + " KAH95/JDS48 0 colonies (re-do)
6. JDS48(BglII, blunted, dp)/ 15 ng 3 colonies
  1 2 3 4 5 6
Insert DNA 6.0 6.0 --- 6.0 6.0 ---
Vector DNA 1.3 1.3 1.3 1.3 1.3 1.3
2x lgn buf (Roche) 8.3 8.3 8.3 8.3 8.3 8.3
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0
dH2O --- --- 6.0 --- --- 6.0
  16.4μL 16.4μL 16.4μL 16.4μL 16.4μL 16.4μL --> R.T./ 30 min.; Add 30 μL Z-DH5α