User:Karmella Haynes/Notebook/BioBrick cloning/2011/02/22

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02/22/11

  • ✓ Gaussia luciferase: check minipreps with NotI digestion
  • ✓ Biobrick PCR: G luc



Gaussia luc minipreps
> Check with NotI digest

Reagent Volume Expected:
Vector = 3030
1-6. G luc/ Addgene22522 = 1433
Hover name
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
NotI 1.0
dH2O 10.5
  15 μL --> 37°C/ ~15 min.

BioBrick PCR
--> Gaussia luciferase from Addgene22522 template

  1. KAH016: (1) Gluc/(X/P PCR)/558 + V0120/(X/P)/~3200 ✓


> PCR
--> Primers: BB_Gluc f/ BB_Gluc r

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; 55°C/ 30 sec; 72 °C/ 1 min.] x30
  • 72 °C/ 3 min.
  • 4 °C/ ∞

> Zymo clean PCR; elute w/ 25 μL dH2O

> Digests (Fermentas FD)

Reagent Volume   PCR digest 02/22/11
30 μL/lane, 1% agarose
Expected = 558 bp
PCR DNA 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. G luc (X/P) ### ### ###

??? -- Nanodrop computer isn't working. Just use max volume insert for 10.0 μL reaction.


> Ligations

Ligation Plate results (lig : neg crtl) 02/23/11
1. G luc(X/P)/558, 8.7 ng + V0120(X/P)/~3200, 25 ng G luc (KAH016) >10:1 (Pick 4)
2. V0120(X/P)/ 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

--> R.T./ ~15 min.; Add to 30 μL DH5α turbo



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