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Team:Newcastle/12 July 2010
From 2010.igem.org
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Revision as of 15:38, 21 July 2010
Monday
Contents |
Aims
The aim of todays Lab session was to perform the following(see summary) to isolate lacI.
Summary:
- Colony PCR
- Streak plate
- Miniprep PCR- of colonies that worked
Equipment
- PCR reagents
- Agar plates
- Gloves
- Wire loop
Colony PCR
Of the 11 plates with colonies grown 7 were used for colony PCR and Streak plating. Colony PCR is less accurate than the mini prep however results can be seen quicker whereas the miniprep has a 1 day wait. The colonies selected selected were satellite colonies because ampicillin degraded???
7 Tubes were labelled 1-7 (+ a control)
The quantities for the PCR are as follows:
|
- 1microlitre Template
- 0.25 microlitre GoTaq Polymerase
- 2.5 microlitre forward primer
- 2.5 microlitre reverse primer
- 1 microlitre dNTP (10molar stock)
- 10.0 microlitre 5times GoTaq buffer
- 32.75 microlitre deionised H20
Note There are 2 possible reasons for red colonies formed:
- Partial digest- rejoins without insert
- with insert
We need to tell the difference between the two.
Note PCR is best when everything is kept on ice!
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Note Melting temperatures of primers is important and can have a big effect on the product formed (Temperature used see Thursday)
Spread Plates
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Tommorrow
We are going to take one of the colonies that have worked and possible a whole plasmid prep.
