PCR

Set up quickly on ice or in a chilled rack. Place imediately in a hot PCR machine.

100ul 25ul Final concentration

10 x PCR buffer 10 ul 2.5 1 x

10 mM dNTP's 2 ul 0.5 0.2 mM

Primer 1 (10 pM/ul) 10 ul 2.5 100 pM

Primer 2 (10 pM/ul) 10 ul 2.5 100 pM

DNA template 10-50 ng 2.5 10-50ng

Taq polymerase (5 U/ul) 1 ul 0.25 0.05 U/ul

Add water to bring to the final reaction volume

Add 2 drops of oil

Standard run: 30 cycles: 94oC for 1 min., 55oC for 2 min and 72oC for 3 min.

Calculate the extension time as Imin/kb.

We have found 1=1=1 to work well for most things.

Hot start method i.e. heating to 940C for 5-10 min. before starting the 30 cycles can be used for templates with secondary structures.

PCR buffer: 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl (pH 8.3).

 

 

SCREENING COLONIES:

Use 25ul reactions for screening colonies.

1. Touch colony with toothpick then dip pick into 30ul TE and touch master plate.

2. Boil sample 5 mins. Spin 5 mins.

3. Add 15ul of above to tubes containing 10 ul of other PCR reagents premixed for a 25 ul rxn.

 

PCR CLEAN UP

1. Remove oil. Add PCR sample to 100ul "direct purification buffer. Vortex.

2. Add 1ml of PCR resin. Mix for 1 min. Apply to syringe barrel inserted into column on vacc-man with vacuum on.

3. Wash with 2 mls of 80% isopropanol. Break vac once. Leave to dry 2 mins on vac-man.

CHECK RESIN IS IN COLUMN AND NOT SYRINGE BARREL

4. Spin column once for 2 mins

5. Leave on bench for 15 mins (NOT LONGER) to allow isopropanol to evaporate.

6. Add 50ul TE at 65C-80C for 1 min

7. Spin for 2 mins

8. Check 5 ul sample on gel.

 

PCR-sequencing

To screen colonies do colony PCR as above with one vector and one insert or two vector oligos. Then do PCR clean up to remove the oligos. To sequence a PCR insert use 1-2ul as template.

1. End label primer- (no need to run a column)

2. Add 3ul of GATor C to colored tubes

3. Mix:

2ul template

2.5 ul end labeled primer done in usual way

1.5 ul 10x vent buffer

1.0 ul 30x triton buffer

H2O to 14 ul

1ul Vent exo-

4. Add 3.2 ul of above to colored tubes. Add 1 drop of oil. Do 20 PCR cycles (1+1+1)

5. Add 4ul stop buffer and heat to 80C for 2 mins

6. Load 2.0 ul