Team:British Columbia/Protocols/Sdm

From 2011.igem.org

(Difference between revisions)
(Created page with "{{Template:Protocols}}")
Line 1: Line 1:
{{Template:Protocols}}
{{Template:Protocols}}
 +
1) Prepare reaction master mix for n+1 reactions, where n is the number of samples on which you wish to use SDM. For a single reaction, use the following reactants in the quantity given.
 +
 +
  RXN Reagent                      1x quantity
 +
 +
  dH2O                            36 uL
 +
  10X Thermopol buffer            5 uL
 +
  dNTP (20 mM)                    1 uL
 +
  FW Primer                        125 ng (primers from IDT diluted to 125 ng/uL, 1 uL/reaction used)
 +
  RE Primer                        125 ng (primers from IDT diluted to 125 ng/uL, 1 uL/reaction used)
 +
  PFU                              2 uL
 +
  MgCl2                            1 uL
 +
  DMSO                            1 uL
 +
 +
2) Aliquot 48 uL master mix into PCR tubes. Remember to keep a water control. Add 50 ng template (diluted to 25 ng/uL, 2 uL/reaction used) per tube to be used for reaction. Add 2 uL dH2O for water control. Be sure to clearly label the PCR tubes!
 +
 +
3) Place PCR tubes in thermocycler, and follow the following cycling conditions.
 +
 +
  Temperature                Time
 +
  95°                        30 seconds
 +
  95°                        30 seconds
 +
  (Tm of primers-5°)          1 minute
 +
  68°                        1-2 minute/KB
 +
  Goto line 2 for 15 more times
 +
  68°                        10 minutes
 +
 +
4) Add 0.5 uL DPN/ reaction and treat at 37° for 60 minutes or overnight to get rid of the template. Run samples on gel to confirm SDM worked.
 +
 +
Troubleshooting notes:
 +
*1 uL PFU was initially used, but 2 uL gave a brighter band on the gel
 +
*10 mM dNTPs were originally used, but there was no product until we increased the concentration to 20 mM.
 +
*First reactions did not use MgCl2 or DMSO, but we found that their addition gave a more consistent, brighter result on the gel.
 +
*PFU was used due to exonuclease activity. Only 16 cycles in total were used to prevent non-specific amplification.
 +
*1 uL of 10 uM/uL primers were initially used, but the reaction did not work until we added the suggested mass.

Revision as of 02:40, 15 August 2011

Team: British Columbia - 2011.igem.org

iGEM Tips for Success
 Things to think about when designing your project and experiments, as well as general safety rules.

Site Directed Mutagenesis
 A molecular biology technique in which a mutation is created at a defined site in a DNA molecule.

Bacterial Standard Operating Protocols
 How to prepare competent cells, transform your construct into competent cells, and express your protein from a lac promoter.

Yeast Standard Operating Protocols
 How to transform your construct into yeast, obtain crude extract for SDS-PAGE, and perform GFP fixation for microscopy and fluorescence-activated cell sorting.

Gas Chromatography-Mass Spectrometry (GC-MS)
 A method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample.

Beetle Transfer Experiments
 Preliminary experiments to probe the efficiency of transferring yeast via beetle vector.

Yeast-Fungi Co-culture Experiments
 Preliminary experiments to investigate the competitive interactions between yeast and the Bluestain Fungus.


1) Prepare reaction master mix for n+1 reactions, where n is the number of samples on which you wish to use SDM. For a single reaction, use the following reactants in the quantity given. 

  RXN Reagent                      1x quantity

  dH2O                             36 uL
  10X Thermopol buffer             5 uL 
  dNTP (20 mM)                     1 uL
  FW Primer                        125 ng (primers from IDT diluted to 125 ng/uL, 1 uL/reaction used)
  RE Primer                        125 ng (primers from IDT diluted to 125 ng/uL, 1 uL/reaction used)
  PFU                              2 uL
  MgCl2                            1 uL
  DMSO                             1 uL

2) Aliquot 48 uL master mix into PCR tubes. Remember to keep a water control. Add 50 ng template (diluted to 25 ng/uL, 2 uL/reaction used) per tube to be used for reaction. Add 2 uL dH2O for water control. Be sure to clearly label the PCR tubes!

3) Place PCR tubes in thermocycler, and follow the following cycling conditions. 

  Temperature                 Time
  95°                         30 seconds
  95°                         30 seconds
  (Tm of primers-5°)          1 minute
  68°                         1-2 minute/KB
  Goto line 2 for 15 more times
  68°                         10 minutes

4) Add 0.5 uL DPN/ reaction and treat at 37° for 60 minutes or overnight to get rid of the template. Run samples on gel to confirm SDM worked.

Troubleshooting notes:

  • 1 uL PFU was initially used, but 2 uL gave a brighter band on the gel
  • 10 mM dNTPs were originally used, but there was no product until we increased the concentration to 20 mM.
  • First reactions did not use MgCl2 or DMSO, but we found that their addition gave a more consistent, brighter result on the gel.
  • PFU was used due to exonuclease activity. Only 16 cycles in total were used to prevent non-specific amplification.
  • 1 uL of 10 uM/uL primers were initially used, but the reaction did not work until we added the suggested mass.
Retrieved from "http://2011.igem.org/Team:British_Columbia/Protocols/Sdm"