User:Karmella Haynes/Notebook/BioBrick cloning/2011/08/07

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08/07/11

  • ✓ Cultures/minipreps: KAH235(2), retransformations DPRE, MV1
  • ✓ Assemblies: PCR inserts for pSB31 cloning



Minipreps
> Check 1-3 with E/S digests (DPRE has PstI sites)
> Check 4 w/ E/P

Reagent Volume Expected:
1,2. KAH235 (E/S) = 2787
3. DPRE (E/P) = 1740
4. MV1 (E/S) = 4102, 1326
File:KAH 080711 gel1.tif
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
SpeI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

--> Mixed up minipreps MV1 and DPRE (3 is MV1 and 4 is DPRE KAH014)
3. MV1 (E/S) = 4742, 686
4. DPRE (E/P) = 3200, 1740 (forgot that I had done s.d. mutagenesis on DPRE to make KAH014, 4/02/10)


Assemblies

  1. KAH222/pSB31: (1) KAH222/(E)/2614 + (9) pSB31/(E dp)/5127
  2. KAH223/pSB31: (2) KAH223/(E)/1537 + "
  3. KAH224/pSB31: (3) KAH224/(E)/1837 + "
  4. KAH232/pSB31: (4) KAH232/(E)/1780 + "
  5. KAH219/pSB31: (5) KAH219/(E)/2422 + "
  6. KAH220/pSB31: (6) KAH220/(E)/1345 + "
  7. KAH221/pSB31: (7) KAH221/(E)/1645 + "
  8. KAH231/pSB31: (8) KAH231/(E)/1588 + "


> PCR
--> Templates: KAH222, 223, 224, 232, 219, 220, 221, 231
--> Primers "Eco insert F1/ r1" match BB prefix/ suffix and add EcoRI to the suffix end

Reagent Volume
DNA (plasmid) 1.0
10 μM primers 1.0
2x GoTaq 25.0
dH2O 23.0

--> BioRad DNA Engine, block A

  • 95°C/ 3 min.
  • [95°C/ 30 sec; 57°C/ 30 sec; 72 °C/ 2 min.] x30
  • 72 °C/ 3 min.
  • 4 °C/ ∞

> Zymo clean PCR; elute w/ 25 μL dH2O


> Digests (Fermentas FD)
--> Specific notes

Reagent Volume   File:KAH 080711 gel2.tif
30 μL/lane, 1% agarose
DNA (plasmid) up to 25 μL
10x buffer 3.0
EcoRI 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH222 (E PCR) 0.728 1.97 36.4
2. KAH223 (E ") 1.064 1.90 53.2
3. KAH224 (E ") 0.944 1.81 47.2
4. KAH232 (E ") 0.862 1.92 43.1
5. KAH219 (E ") 0.803 1.78 40.2
6. KAH220 (E ") 1.182 1.87 59.1
7. KAH221 (E ") 0.743 1.79 37.2
8. KAH231 (E ") 0.964 1.92 48.2
9. pSB31 (E ") 0.991 1.89 49.5


> Dephosphorylation (Roche)
--> pSB31 (E)

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) 08/08/11
1. KAH222(E PCR)/2614, 20 ng + pSB31(E dp)/5127, 20 ng KAH222/pSB31 >10:1 (Pick 3)
2. KAH223(E PCR)/1537, 12 ng + " KAH223/pSB31 >10:1 (Pick 3)
3. KAH224(E PCR)/1837, 12 ng + " KAH224/pSB31 >10:1 (Pick 3)
4. KAH232(E PCR)/1780, 14 ng + " KAH232/pSB31 >10:1 (Pick 3)
5. KAH219(E PCR)/2422, 19 ng + " KAH219/pSB31 >10:1 (Pick 3)
6. KAH220(E PCR)/1345, 11 ng + " KAH220/pSB31 >10:1 (Pick 3)
7. KAH221(E PCR)/1645, 13 ng + " KAH221/pSB31 >10:1 (Pick 3)
8. KAH231(E PCR)/1588, 12 ng + " KAH231/pSB31 >10:1 (Pick 3)
2. pSB31(E dp)/ 20 ng  
  1 2 3 4 5 6 7 8 9
Insert DNA 0.6 0.3 0.3 0.3 0.5 0.3 0.4 0.3 ---
Vector DNA 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8
2x lgn buf (Roche) 5.0 5.0 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 1.0 1.0
dH2O 2.6 2.9 2.9 2.9 2.7 2.9 2.8 2.9 3.2
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL

--> Add to 30 μL DH5α Turbo; plate on 100 μg/mL Amp