Difference: ProtocolsReagentsPfuSso7d (20 vs. 21)

Revision 212018-06-08 - JeffreyBarrick

Line: 1 to 1
 
META TOPICPARENT name="ProtocolsReagentRecipes"

Protocol for harvesting Pfu-Sso7d polymerase

Line: 47 to 47
 
  • 0.2% Tween20
  • 2 mM DTT (add immediately before using for storage)
Changed:
<
<
IMPORTANT: Add fresh DTT immediately before using a batch of storage buffer to store newly purified enzyme. Your fresh DTT should be newly dissolved from powder or from a 1 M stock that you store frozen at –20C to avoid oxidation. We have observed rapid loss of function of enzyme when enzyme is diluted in old storage buffer that had DTT added and was then stored at room temperature.
>
>
Warning, important IMPORTANT: Add fresh DTT immediately before using a batch of storage buffer to store newly purified enzyme. Your fresh DTT should be newly dissolved from powder or from a 1 M stock that you store frozen at –20C to avoid oxidation. We have observed rapid loss of function of enzyme when enzyme is diluted in old storage buffer that had DTT added and was then stored at room temperature.
 

Protein Expression

Line: 64 to 64
 
  • Collect cells by centrifugation. Conditions as follows: 4C, at 10,000 x g for 15 mins.
  • Resuspend each cell pellet with 3 mL of lysis buffer and combine tubes together, mix well using pipette.
  • French press; use the full cell holding (10 mL - 35 mL) and 1500 psi pressure.
Changed:
<
<
  • Collect and reintroduce into french press 1x.
>
>
  • Collect and reintroduce into French press 1x.
 
  • Heat denature at 70C for about 15 mins.
  • Spin down, 10,000 x g for 30 mins.
  • Syringe filter the supernatant (0.22 m filter).
Line: 79 to 79
 
  • Wash with 5× (column volume) of wash buffer.
  • Elute with 3 mL of elution buffer and collect all 3 mL of elution.
Added:
>
>
Warning, important NOTE: This purification protocol does not perfectly purify the polymerase from all other E. coli proteins. Expect your protein sample to still be heterogeneous if you run it on a gel. Don't worry, it will have sufficient purity for DNA amplification. The heating step at 70C during harvesting helps precipitate many E. coli proteins.
 

Dialysis:

  • Place dialysis cassette into storage buffer for 2 mins.
  • Remove top and load dialysis cassette with enzyme sample using a pipette or syringe.
 
This site is powered by the TWiki collaboration platform Powered by Perl This site is powered by the TWiki collaboration platformCopyright ©2021 Barrick Lab contributing authors. Ideas, requests, problems? Send feedback