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Revision as of 21:17, 13 July 2010 (view source) RachelBoyd (Talk | contribs) (New page: '''Monday''' ===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 th...) ← Older edit		Revision as of 21:23, 13 July 2010 (view source) RachelBoyd (Talk | contribs) (→Colony PCR) Newer edit →
Line 32:		Line 32:
	* 10.0 microlitre 5times GoTaq buffer		* 10.0 microlitre 5times GoTaq buffer
	* 32.75 microlitre deionised H20		* 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.

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Revision as of 21:23, 13 July 2010

Monday

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

Equipement

• 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.