Manual KOD PCR Setup

Overview of Manual KOD setup

Preparation:

• The stock primers-forward and reverse-will need to be combined and diluted. This is the Oligo plate to be used for further experiments.
• Select a round bottom 96 well plate to use for the combination of primers.
• Using the SAMI program on the computer, select the "Print and Apply" function. Label the round bottom plate "F/R Oligos (Plate ID*) Today's Date".
• Locate a drilled bottom 96 well plate. These are usually kept in the organizer to the right of the sink with the reagent reservoirs.
• Place a PCR plate (flexible and non skirted- found in the cabinet) in the drilled bottom 96 well plate. This offers stability to the PCR plate when it is in the Multimek and keeps the PCR plate at the right height.
• As a reminder, ice will be required for PCR, and all PCR components shoµld be kept on ice, in advance. Regarding the addition of PCR components, it shoµld be noted that KOD will be added to PCR mastermix last.

Procedure:

• PCRfc1 is the program on the Multimek that will combine and dilute the stock primers.
• PCRFC3 will transfer 10ml of the "F/R Oligo" plate to the supported PCR plate (from step 5).

Cleanup:

• The stock primers are stored in the freezer at -20°C -or if they will not be needed for an extended period of time, they can be stored in the -70°C freezer on the service level.
• The "F/R Oligo" plate is stored at 4°C in the fridge.
• The supported PCR plate is ready for "Manual KOD PCRx setup".

• *Plate ID Examples: GTL01, BCer13, Styp01
  
  
• Next Step: Manual KOD PCRx

Multimek Method PCRFC1

Multimek Method PCRFC3

Overview of smKOD Rxn Setup

Preparation:

• KOD Kits are kept at -20°C. An ice bucket will be needed to prepare the reaction mix. 
• All components must be kept on ice throughout the experiment. 
• In an ice bucket, use the components from the KOD Kit to prepare the mix as follows for one plate: 
• Water                                  2880 µl (3 x 960µl) 
• 10x KOD Buffer                    540 µl 
• dNTP's                                  540 µl 
• MgSO4                                  216 µl 
• KOD Polymerase                     90 µl     (Add the KOD Polymerase last) 
• Genomic DNA                         72 µl     (500-1000 ng total)=8µl of gDNA + 64µl of water 
• gDNA is kept in the freezer, -20°C , until it is diluted with TE, then it is stored in at 4°C. 
• Mix the solution well.

Procedure:

• Transfer 40 µl of KOD Reaction Mix to the supported PCR plate using smKOD method on the BioMek. 
• Centrifuge BRIEFLY to insure all liquid is at the bottom of the wells. 
• The PCR plate is now ready to be placed in the thermocycler machine. 
• Be sure to get the proper conditions for your organism: 
• PCR program KODI works for Shewanella and Geobacter: 
•                 (Some Geobacter targets may require touch down PCR) 
• Denature             95°C for 3 minutes 
• Cycle                  95°C for 40 seconds 
•                            53°C for 40 seconds 
•                            72°C for 1.5 minutes (depends on anticipated product length) 
• Polish                  72°C for 5-10 minutes
  
  
• Next Step: Manual Qiagen PCR Purification

smKOD Rxn Setup