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From 2010.igem.org

MAIN STEPS / TIMETABLE
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- Restriction Digestion [1-16 h]
- PCR purification [1 h]
- Agarose Gel Electrophoresis [2 h]

MATERIALS
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- Heat Block
- Spin
- Nanodrop

- Nuclease Free Water
- Vector
- Buffers
- Restriction Enzymes ( EcorI, XbaI, SpeI, PstI)

- 0.2 mL eppendorf
- P10 micropipette
- Parafilm
- Ice
- Ice box

CHECK-LIST PROCEDURE
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Experiment Preparation
- Nuclease free water and eppendorfs are autoclaved (1 day before)
- Heat block is set to 37 C

Preparing the Total Mix (for 20 uL total mix)
- Add uL water nuclease free to 0.2 mL PCR tube
- Add 2 uL appropriate 10x buffer(depending on enzyme type) (thawed before)
- Add uL DNA solution to the tube (ugr)
- Add 15 unit restriction enzyme to the tube
- Spin the tube for 5 sec
- Incubate the tube for h at 37 C at heatblock

Enzyme Inactivation (EcorI and XbaI)
- Spin the enzyme solution at 100 g for 15 sec
- Incubate at 65 C for 15 min at heatblock
- Store at -20 C in the freezer

Enzyme Inactivation (SpeI and PstI)
- Spin the enzyme solution at 100 g for 15 sec
- Inactivate the enzyme with Fermentas PCR Purification kit
- Store at -20 C in the freezer

Enzyme Inactivation (Fastdigest NotI)
- Spin the enzyme solution at 100 g for 15 sec
- Incubate at 80 C for 5 min at heatblock
- Store at -20 C in the freezer

Agarose Gel Electrophoresis
- Look at procedure for agarose gel electrophoresis
- Load 2 uL sample for confirmation of the restriction
- Load whole sample for insert extraction

Double Digestion

>>> EcorI-SpeI Double Digestion >>>
- Buffer R
- 4-fold excess of BcuI
- EcoRI
- Incubate at 37C
>>> XbaI - PstI Double Digestion
- Buffer O
- PstI
- 4-fold excess of XbaI
- Incubate at 37C
>>> EcorI - PstI Double Digestion
- Buffer O
- PstI
- EcorI
- Incubate at 37C