Endy:Colony PCR protocol

Solutions/reagents: Thermo polymerase bufferdNTPSForward primerReverse primerTaq or Vent PolymeraseTemplate suspensionH2O Equipment: Thermocycler</li>Reaction tubes</li></ul> Steps: <ol> <font color = "#800517">Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 ?l sterile water. </li> <font color = "#800517">Store the colony resuspension at 4°C so you can start cultures if necessary (should be OK for a couple days, if you need it to last longer you should use an Index plate. </li>   Reaction mix  Set up a reaction in reaction tube (1) as follows on ice:  </li>   PCR protocol  Program a standard thermocycler to run the reaction using the following parameters: Initial denaturation Denature: <font color=#357EC7>95°C , <font color=#357EC7>6 mins </li></ul>Thermocycling No. of cycles: <font color=#357EC7>35 </li>Denature: <font color=#357EC7>95°C , <font color=#357EC7>30 secs </li>  Anneal: <font color=#357EC7>53°C , <font color=#357EC7>30 secs </li> Elongate: <font color=#357EC7>72°C , <font color=#357EC7>60 secs </li></ul>TerminationElongate: <font color=#357EC7>72°C , <font color=#357EC7>10 mins </li>Hold: <font color=#357EC7>4°C , until removed from machine </li></ul><font color = "#800517">For long amplicons, elongation time = 1 minute + 2.5 s per 100bp. <font color = "#800517">For shorter amplicons, under ~1kb, this can be shortened judiciously. </li> </ol> TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 1 hr, 28 mins