Team:UC Davis/Protocols

From 2011.igem.org

(Difference between revisions)
Line 108: Line 108:
*.15ul PFU
*.15ul PFU
*30ul dH2O
*30ul dH2O
 +
 +
=Minipreps=
 +
 +
====Materials====
 +
* Liquid culture
 +
* Miniprep kit (QIAprep Spin Miniprep Kit)
 +
 +
====Procedure====
 +
* Centrifuge liquid culture of cells.
 +
* Discard the supernatant.
 +
* Resuspendd the pelleted cells in 250 uL of Buffer P1 and transfer to a microcentrifuge tube.
 +
* Add 250 uL of Buffer P2.
 +
**Invert 4-6 times until the solution become clear.
 +
* Add 350 uL Buffer N3.
 +
** Invert 4-6 times.
 +
* Centrifuge for 10 minutes at 17900xg.
 +
* Apply the supernatant to a spin column.
 +
** Centrifuge for 1 minute and discard the flow-through.
 +
* Wash the spin column with 0.5 ml Buffer PB.
 +
** Centrifuge for 1 minute and discard the flow through.
 +
* Wash the spin column with 0.75 ml Buffer PE.
 +
** Centrifuge for 1 minute and discard the flow through.
 +
* Centrifuge an additional 1 minute to remove residual wash buffer.
 +
* In a clean 1.5 ml microcentrifuge tube, elute DNA with 50 ul Buffer EB.
 +
* Centrifuge for 1 minute and collect the flow-through.

Revision as of 20:22, 29 June 2011

Our Sponsors

Start a Family

Got a favorite BioBrick? Check our our process for expanding basic parts into part families.

Criteria

View our judging criteria for iGEM 2011 here.


Restriction Enzyme Double Digest

Materials

  • 22 uL dH2O
  • 1 uL BSA
  • 5 uL Buffer
  • 20 uL Template
  • 1 uL Enzyme 1
  • 1 uL Enzyme 2

Buffer Compatibility Chart

1 2 3 4
EcoRI 100 100 100 100
SpeI 75 100 25 100
PstI 75 75 100 50
NheI 100 100 10 100
XbaI 0 100 75 100

Procedure

  • Mix reactants thoroughly.
  • Place at 37 C for 3 hours.
  • Increase to 80 C for 20 minutes to kill enzymes (some enzymes need only a 65 C heatkill, check enzyme).
  • Run on a gel and extract product.

Gel Extraction/Purification Procedure

Materials

  • GeneJET Gel Extraction Kit
  • Binding Buffer (1 uL for every mg of agarose gel)
  • 700 uL of Wash Buffer
  • 50 uL of Elution Buffer

Procedure

  • Add the binding buffer to the gel slice in a microcentrifuge tube.
  • Incubate the gel mixture at 50-60 °C for 10 minutes (until melted).
  • Transfer the solution to a GeneJET purification column.
  • Centrifuge for 30-60 seconds at 12000 x g and discard the flow through.
  • Add Wash Buffer and centrifuge for 1 minute.
  • Discard flow through, then centrifuge empty column for 1 minute.
  • Transfer the column into a fresh 1.5 ml microfuge tube.
  • Add Elution Buffer.
  • Centrifuge for 1 minute and collect the flow-through.

Transformations

Materials

  • Competent cells
  • DNA template
  • 800 uL of LB
  • LB+antibiotic plates

Procedure

  • Thaw competent cells on ice.
  • Transfer 50 uL of competent cells to chilled falcon tubes.
  • Add 1 uL of template to cells (2.5 uL if dilute).
  • Incubate on ice for 30 minutes.
  • Heat schock in 42 °C water bath for 90 seconds.
  • Immediately place back onto ice for 2 minutes.
  • Add 800 uL of LB to each tube.
  • Incubate at 37 °C for 1 hour.
  • Place 200 uL of the transformed cells on plates containing LB and the appropriate antibiotic.
  • Incubate overnight at 37 °C.

Liquid Cultures

Materials

  • LB
  • Plated colonies of cells
  • Antibiotic stock

Procedure

  • In a sterile environment, add 4 mL of LB to each falcon tube.
  • Add the appropriate amount of antibiotic.
    • for carb, add 8 uL.
  • With a tip, scoop a colony and place it in the falcon tube.
  • Incubate overnight at 37 °C.

PCR

Mix

  • 10ul Q solution
  • 5ul 10x buffer
  • 1.25ul DNTPs
  • 1ul Forward primer
  • 1ul Reverse primer
  • 1ul Template
  • .3ul Taq
  • .15ul PFU
  • 30ul dH2O

Minipreps

Materials

  • Liquid culture
  • Miniprep kit (QIAprep Spin Miniprep Kit)

Procedure

  • Centrifuge liquid culture of cells.
  • Discard the supernatant.
  • Resuspendd the pelleted cells in 250 uL of Buffer P1 and transfer to a microcentrifuge tube.
  • Add 250 uL of Buffer P2.
    • Invert 4-6 times until the solution become clear.
  • Add 350 uL Buffer N3.
    • Invert 4-6 times.
  • Centrifuge for 10 minutes at 17900xg.
  • Apply the supernatant to a spin column.
    • Centrifuge for 1 minute and discard the flow-through.
  • Wash the spin column with 0.5 ml Buffer PB.
    • Centrifuge for 1 minute and discard the flow through.
  • Wash the spin column with 0.75 ml Buffer PE.
    • Centrifuge for 1 minute and discard the flow through.
  • Centrifuge an additional 1 minute to remove residual wash buffer.
  • In a clean 1.5 ml microcentrifuge tube, elute DNA with 50 ul Buffer EB.
  • Centrifuge for 1 minute and collect the flow-through.
Retrieved from "http://2011.igem.org/Team:UC_Davis/Protocols"