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Revision as of 15:36, 21 July 2010

Monday

Contents 1 Aims 2 Equipment 3 Colony PCR 4 Spread Plates 5 Tommorrow

Aims

The aim of todays Lab session was to perform the following(see summary) to isolate lacI.

Summary:

1. Colony PCR
2. Streak plate
3. 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:

1. Partial digest- rejoins without insert
2. with insert

We need to tell the difference between the two.

Note PCR is best when everything is kept on ice!

[[Image:P7120427.JPG|thumb]right]

Note Melting temperatures of primers is important and can have a big effect on the product formed (Temperature used see Thursday)

Spread Plates

Tommorrow

We are going to take one of the colonies that have worked and possible a whole plasmid prep.