Team:Newcastle/12 July 2010

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:

• 1µl Template
• 0.25µl GoTaq Polymerase
• 2.5µl forward primer
• 2.5µl reverse primer
• 1µl dNTP (10molar stock)
• 10.0µl 5times GoTaq buffer
• 32.75µl 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!

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 possibly a whole plasmid prep.