User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/17

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07/17/11

  • ✓ Cultures/minipreps: KAH219-224 (2 each); p65 Biobrick (clone #3, two new preps from streak)
  • ✓ Assemblies: KAH119-224 into pSB31 (for mouse ES cell expression, Manching Ku)



Minipreps
> Check with E/P digests

Reagent Volume Expected:
Gel 1
1,2. KAH219 = 2422
3,4. KAH220 = 1345
5,6. KAH221 = 1645
7,8. KAH222 = 2614
9,10. KAH223 = 1537
11,12. KAH224 = 1837

Gel 2
1,2. p65 Biobrick clone #3 = 783
3. p65 mut1 template = 470, 313
File:KAH 071711 gel1.tif
15 μL/lane; 1% agarose

File:KAH 071711 gel2.tif
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.

--> p65 worked! Combine preps 1/2. Order sequencing rxn. tomorrow.

Sequencing results (7/18/11)

  1. KAH017v2-3, P0001: good
  2. KAH017v2-3, P0002: good



Assemblies

  1. KAH219/pSB31: (1) KAH219/(E/S blunt)/2422 + pSB31/(E blunt dp)/5127 ✓
  2. KAH220/pSB31: (2) KAH220/(E/S blunt)/1345 + "
  3. KAH221/pSB31: (3) KAH221/(E/S blunt)/1645 + "
  4. KAH222/pSB31: (4) KAH222/(E/S blunt)/2614 + "
  5. KAH223/pSB31: (5) KAH223/(E/S blunt)/1537 + "
  6. KAH224/pSB31: (6) KAH224/(E/S blunt)/1837 + "

> Digests (Fermentas FD)

Reagent Volume   File:KAH 071711 gel3.tif
30 μL/lane, 1% agarose
DNA (plasmid) 25.0
10x buffer 3.0
EcoRI 1.0
SpeI 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


> Measure concentration

Sample OD260 260/280 ng/μL
1. KAH219 (E/S) 0.267 2.17 13.4
2. KAH220 (E/S) 0.518 2.01 25.9
3. KAH221 (E/S) 0.493 1.79 24.7
4. KAH222 (E/S) 0.439 1.97 22.0
5. KAH223 (E/S) 0.256 2.19 12.8
6. KAH224 (E/S) 0.465 2.07 23.3


> Blunting (NEB Quick Blunting kit)

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.; deactivate @ 70°C/ 10 min.

--> Use max. volume for all inserts

  1. KAH219 [final] = 10.2 ng/μL
  2. KAH220 [final] = 19.7 ng/μL
  3. KAH221 [final] = 18.8 ng/μL
  4. KAH222 [final] = 16.7 ng/μL
  5. KAH223 [final] = 9.7 ng/μL
  6. KAH224 [final] = 17.7 ng/μL


> Ligations

Ligation Plate results (lig : neg crtl) 01/23/11
1. KAH160(E/S blunt)/2422, 24 ng + pSB31(E blunt dp)/5127, 25 ng KAH219/pSB31 2:1 (Pick 4)
2. KAH160(E/S blunt)/1345, 13 ng + " KAH220/pSB31 3:1 (Pick 4)
3. KAH160(E/S blunt)/1645, 16 ng + " KAH221/pSB31 4:1 (Pick 4)
4. KAH160(E/S blunt)/2614, 25 ng + " KAH222/pSB31 3:1 (Pick 4)
5. KAH160(E/S blunt)/1537, 15 ng + " KAH223/pSB31 2:1 (Pick 4)
6. KAH160(E/S blunt)/1837, 18 ng + " KAH224/pSB31 5:1 (Pick 4)
7. pSB31(E blunt dp)/ 25 ng  
  1 2 3 4 5 6 7
Insert DNA 2.5 1.0 1.0 1.5 1.5 1.0 ---
Vector DNA 1.0 1.0 1.0 1.0 1.0 1.0 1.0
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O 0.5 2.0 2.0 1.5 1.5 2.0 3.0
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL

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