User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/10: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 72: Line 72:
|}
|}


Dilutions
{| {{table}} border="1" cellspacing="3" <!-- Dilution table -->
|- valign="top"
| bgcolor=#cfcfcf | DNA
| bgcolor=#cfcfcf | length (bp)
| bgcolor=#cfcfcf | ng/μL
| bgcolor=#cfcfcf | Vol.
| bgcolor=#cfcfcf | + dH<sub>2</sub>O
|-
| pSB1A3 || 2000 || 77.4 || 7.7 || 12.3
|-
| hPCD || 186 || 17.0 || 2.1 || 17.8
|-
| BL01 || 2520 || 66.9 || 11.7 || 8.3
|}
* Golden Gate Reactions
# hPCD + BL01 + pSB1A3
# pSB1A3
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
|- valign="top"
| 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
|-
| dH<sub>2</sub>O || 5.25 || 7.25
|-
| &nbsp; || 10.0 || 10.0
|}
Thermal cycler
* [45°C, 2 min.; 16°C, 5 min.] x25
* 60°C, 20 min.
* 80°C, 20 min.




Revision as of 22:01, 10 January 2013

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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


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
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, 3 min.; 95°C, 3 min.; 95°C, 3 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

DNA length (bp) ng/μL Vol. + dH2O
pSB1A3 2000 77.4 7.7 12.3
hPCD 186 17.0 2.1 17.8
BL01 2520 66.9 11.7 8.3


  • 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 cycler

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



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/01/10&oldid=666699"