IGEM:IMPERIAL/2006/LabCalendar/2006-7-12: Difference between revisions
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Revision as of 01:06, 17 July 2006
John Sy
Preparation for digestion of bacteria to isolate DNA for gel electrophoresis. We have identified 4 colonies on our plate containing our part I13273 which are potential cells with our desired DNA. However, only colonies 3 and 4 produced any bacteria. Media containing colonies 1 and 2 were still clear after leaving overnight.
Followed standard procedure for digestion (miniprep)
Formula for Restriction Enzyme addition after miniprep
- 1 uL EcoRI
- 1 uL PstI
- 2 uL Buffer 0 (orange label)
- 6 uL ultrapure water
- 10 uL DNA sample
Heat in waterbath at 37C for approximately 30 minutes
Formula to make gel for electrophoresis
- 0.2 g agarose in 20 mL 1x TAE
- Place in microwave for about 1 minute to dissolve
- Allow to cool to about 60C (warm enough to hold by hand)
- add 1 uL ethidium bromide
- pour into plates to make gels
Formula to prepare digested DNA for electrophroesis
- Add 4 uL loading buffer containing RNAase
- Transfer all 24 uL into one well
- Run electrophoresis at about 80V, 0.5A
Results from digest
We seem to have obtained two different bands from the same restriction enzyme and DNA from two different wells. This is not what we expected. Looking at webcutter, we discover that our DNA part I13273 is not cut by either of the restriction enzymes. Dr. Mann suggests that the different banding may be due to supercoiling of the DNA.
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