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).
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.
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
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