From 2011.igem.org
Restriction Enzyme Double Digest
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
{| border="1"
!
!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.
Ligations
Materials
* Digested vector
* Digested insert
* Water
* T4 DNA ligase.
* T4 DNA ligase buffer.
Procedure
* Mix these materials in the amounts determined by the reaction volume calculator.
[[media:UC_Davis_Reaction_Volume_Calculator.xls]]
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