User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/22

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01/22/11

  • ✓ Re-do ligation: SP1AB w.t./V0120
  • ✓ Miniprep: pSB31 (2)
  • ✓ Assemblies: KAH160, 165, 170 into pSB31



Re-do ligations from 01/21/11

> Dephosphorylation (Roche)

  1. V0120 X/S vector
Reagent Volume
DNA (clean digest) up to 17 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


> Ligations

Ligation Plate results (lig : neg crtl) 01/23/11
1. SP1AB w.t.(X/S)/1617, 25 ng + V0120(X/S)/~3200, 25 ng SP1AB w.t./V0120 5:1 (Pick 6)
2. V0120(X/S)/ 25 ng  
  1 2
Insert DNA 3.0 ---
Vector DNA 1.0 1.0
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O --- 3.0
  10 μL 10 μL


--> ~1 hr./ r.t.; Add to 30 μL DH5α turbo



Assemblies

  1. KAH160/pSB31: KAH169/(E/S blunt)/1147 + pSB31/(E blunt)/5127
  2. KAH165/pSB31: KAH165/(E/S blunt)/991 + "
  3. KAH170/pSB31: KAH170/(E/S blunt)/955 + "

> Digests (Fermentas FD)
--> Digest pSB31 vector w/ EcoRI; 2 rxns
--> Do not gel purify

Reagent Volume  
DNA (plasmid) 20.0
10x buffer 3.0
EcoRI 1.0
dH2O 5.0
  30 μL --> 37°C/ ~30 min.

--> Zymo clean EcoRI cut vector; elute w/ 25 μL dH2O


> 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. KAH160 (max. vol.) [final] = 2.7 ng/μL
  2. KAH165 (max. vol.) [final] = 6.9 ng/μL
  3. JDS170 (max. vol.) [final] = 4.6 ng/μL
  4. pSB31 (max. vol., measure after cleaning)

--> Clean vector DNA using Zymo DNA Clean & Concentrator; elute w/ 20 μL dH2O


> Measure concentration

Sample OD260 260/280 ng/μL
1. pSB31 (E blunt) 2.194 1.83 109.7


> Dephosphorylation (Roche)
--> pSB31 (E blunt); 3 rxns

Reagent Volume
DNA (clean) up to 17 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


> Ligations

Ligation Plate results (lig : neg crtl) 01/23/11
1. KAH160(E/S blunt)/1147, 11 ng + pSB31(E blunt dp)/5127, 25 ng KAH160/pSB31 5:1 (Pick 6)
1. KAH160(E/S blunt)/991, 10 ng + " KAH160/pSB31 5:1 (Pick 6)
1. KAH160(E/S blunt)/955, 9 ng + " KAH160/pSB31 5:1 (Pick 6)
2. pSB31(E blunt dp)/ 25 ng  
  1 2 3 4
Insert DNA 3.0 3.0 3.0 ---
Vector DNA 1.0 1.0 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 --- --- --- 3.0
  10 μL 10 μL 10 μL 10 μL

--> ~1 hr./ r.t.; Add to 30 μL DH5α turbo