User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/14

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07/14/11

  • ✓ Cultures/ minipreps: miR-luc sensor "177/MV8" set (3 each, 24 total)
  • ✓ SOEing & cloning: mutate second PstI site in p65 biobrick



Minipreps
> Check with ApaLI digest

Reagent Volume Expected:
MV8 bands = 1243, 1135, 288
1-3. 186-177/MV8 = 3860*, 2772**, 711
4-6. 184-177/MV8 = 4026*, 2772**, 711
7-9. 187-177/MV8 = 4104*, 2772**, 711
10-12. 188-177/MV8 = 3959*, 2772**, 711
13-15. 189-177/MV8 = 4131*, 2772**, 711
16-18. 190-177/MV8 = 4203*, 2772**, 711
19-21. 191-177/MV8 = 4058*, 2772**, 711
22-24. 192-177/MV8 = 4296*, 2772**, 711
*insert + 234 (vector) + 1601 (177 frag)
**last 177 frag + 1655
Miniprep digests 7/14/11
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.



PCR SOEing
> Site directed mutagenesis failed twice for second PstI site in p65 biobrick
> Step 1: megaprimers from p65 (single mut) template

  1. Amplicon 1: BB_p65 f1/ mut_p65 3
  2. Amplicon 2: mut_p65 2/ BB_p65 r1


> PCR step 1

Reagent Volume Expected:
1. Amplicon 1 = 484
2. Amplicon 2 = 326
PCR 7/14/11
5 μL/lane; 1% agarose
DNA(plasmid) 1.0
10 μM primer 1 1.0
10 μM primer 2 1.0
2x GoTaq green 25.0
dH2O 22.0
  50 μL

--> BioRad Block A

  • 95°C/ 3 min.
  • [95°C/ 30 sec., 55°C/ 30 sec., 72°C/ 30 sec.] x35
  • 72°C/ 3 min.
  • 4°C/ ∞

--> Clean bands 1 and 2 using Zymo clean & concentrator; elute w/ 20 μL dH2O


> PCR step 2

Reagent Volume Expected:
783 bp
PCR2 7/14/11
10 μL/lane; 1% agarose
amplicon 1 (clean) 5.0
amplicon 2 (clean) 5.0
2x GoTaq green 25.0
dH2O 15.0
  50 μL

--> BioRad Block A

  • 95°C/ 3 min.
  • [95°C/ 30 sec., 55°C/ 30 sec., 72°C/ 30 sec.] x35
  • 72°C/ 3 min.
  • 4°C/ ∞

--> Zymo clean & concentrator; elute w/ 25 μL dH2O


> Digest (Fermentas FD)

Reagent Volume   SOEing PCR digest 7/14/11
30 μL/lane, 1% agarose
DNA (clean PCR2) 25.0
10x buffer 3.0
XbaI 1.0
PstI 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. p65 PCR2 (X/P) 0.185 2.41 9.2


> Ligations

Ligation Plate results (lig : neg crtl) 07/15/11
1. p65 PCR2(X/P)/783, 12 ng + V0120(X/P)/~3200, 25 ng p65 ~10:1, 8 colonies (Pick 4)
2. V0120(X/P)/ 25 ng 1 colony
  1 2
Insert DNA 1.3 ---
Vector DNA 0.7 0.7
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 2.0 2.0
  10 μL 10 μL

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




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