User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/10

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PCR
PCR
* 95°C, 3 min.
* 95°C, 3 min.
-
* [95°C, 3 min.; 57°C, 3 min.; 72°C, 3 min.] x35
+
* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
* 72°C, 3 min.
* 72°C, 3 min.
* 4°C, ∞
* 4°C, ∞
Line 74: Line 74:
-
Dilutions
+
Dilutions: to final concentration of 20 fmol/μL
{| {{table}} border="1" cellspacing="3" <!-- Dilution table -->
{| {{table}} border="1" cellspacing="3" <!-- Dilution table -->
|- valign="top"
|- valign="top"

Current revision

Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

01/10/13

  • Violacein: successful growth; re-streak
  • Golden gate/ transformation: transform DH5α-Turbo with gg assemblies from 1/09/13
  • Golden gate trial 2: new PCR primers


Violacein

  • Plate 1: Kan plasmid
  • Plate 2: Amp plasmid (violacein exp.)


Golden gate trial 1

  • Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar
  • Colonies: 7 on assembly plate, 9 on vector-only control. Pick 4 and make 2 mL liquid cultures.


Golden gate trial 2

  • Re-designed primers to include "handle" preceding the BsmBI sites and a spacer between BsmBI and a 4 bp overlap region
Reagent Volume Expected:
1. pSB1A3 = ~2000
2. hPCD = 186
3. BL01 = 2520 		Hover name
10 μL/lane; 1% agarose; Ladder
DNA(plasmid) 0.2 μL
primer 1 1.0
primer 2 1.0
2x GoTaq 25.0
dH2O 22.8
  50.0 μL

PCR

  • 95°C, 3 min.
  • [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞


Purification

  • Clean with Zymo DNA c&c; elute w/ 20 μL dH2O
  • Measure [DNA]
Sample OD 260 260/280 ng/μL
1. pSB1A3 0.126 1.888 77.357
2. hPCD 0.069 2.192 17.025
3. BL01 0.121 1.898 66.878


Dilutions: to final concentration of 20 fmol/μL

DNA length (bp) ng/μL Vol. + dH2O
pSB1A3 2000 77.4 6.7 13.3
hPCD 186 17.0 2.8 17.2
BL01 2520 66.9 9.8 10.2


Golden Gate Reactions

  1. hPCD + BL01 + pSB1A3
  2. pSB1A3
Reagent 1 2
gg2 pSB1A3 1.0 1.0
gg3 hPCD 1.0 ---
gg4 BL01 1.0 ---
10x ligase buffer 1.0 1.0
NEB T4 lgase 0.25 0.25
BsmBI 0.5 0.5
dH2O 5.25 7.25
  10.0 10.0

Thermal cycling

  • [45°C, 2 min.; 16°C, 5 min.] x25
  • 60°C, 20 min.
  • 80°C, 20 min.
  • 4°C, ∞



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