IGEM:IMPERIAL/2006/LabCalendar/2006-7-11: Difference between revisions
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*Solution 1 | *<u>Solution 1</u> | ||
*90 g glucose | |||
*10 mL 0.5M EDTA | |||
*6.25 mL 2M Tris pH 8 | |||
*Make up to 500 mL | |||
<u>Solution 2</u> | |||
*50 mL 10% SDS | |||
*20 mL 5M NaOH | |||
*Make up to 500 mL | |||
<u> Solution 3</u> | |||
*147 g Potassium acetate | |||
*57.5 mL glacial acetic acid | |||
*Make up to 500 mL | |||
* | |||
147 g Potassium acetate | |||
57.5 mL glacial acetic acid | |||
Make up to 500 mL | |||
Revision as of 04:00, 20 July 2006
Miniprep- Prepare Plasmid DNA for electrophoresis
Follow miniprep protocol to prepare plasmid DNA samples for gel electrophoresis. Protocol for bacteria digestion:
- Transfer 1.5 mL of culture into eppendorf
- Spin for 20 sec and remove supernatant
- Add 100 uL of solution 1 and vortex to resuspend
- Add 200 uL of solution 2
- Add 150 uL of solution 3
- Mix by inverting, white ppt appears
- Spin for 3 minutes
- Pour supernatant in new labeled tube, add 1 mL of ethanol and mix
- Spin for another 3 minutes and remove supernatant
- Spin to remove resitudal liquid and then resuspend in 50 uL of MiliQ (ultrapure water) (1 sec on vortex, don't resuspend pellet)
- Spin for 1 minute
- Read to digest!
- Solution 1
- 90 g glucose
- 10 mL 0.5M EDTA
- 6.25 mL 2M Tris pH 8
- Make up to 500 mL
Solution 2
- 50 mL 10% SDS
- 20 mL 5M NaOH
- Make up to 500 mL
Solution 3
- 147 g Potassium acetate
- 57.5 mL glacial acetic acid
- Make up to 500 mL
Digest using enzymes EcorI (1microlitre) and PS21 (1 microlitre) to digest 10 microlitres of DNA.
Orange buffer (2microlitres) optimal for both restriction enzymes' activity.
6 microlitres of distilled water added to achieve appropriate dilutions.
Leave at 37C (water bath) for 45 minutes.
