Overview of Procedures for Biology Project Amplifying the GAPC Gene from Diverse Plant Species

Data Collection on 1-17 Nucleic Acid Extraction from Plants –Grind and lyse plant tissue to release DNA –Purify DNA away from contaminating RNA and protein by using a silica column

Data Collection on 1-17 Set up PCR Round 1 Prepare a series of tubes as negative and positive controls and experimental samples with plant DNA Instructor will add primers Thermal cycler will make temperature changes Samples will be held in freezer until next session

Data Collection on 1-21 Set up PCR Round 2 –Treat selected samples from Round 1 with exonuclease to remove primers –Inactivate the exonuclease!

Data Collection on 1-21 Set up PCR Round 2 –Prepare a series of tubes as negative and positive controls and experimental samples with plant DNA –Instructor will add primers –Thermal cycler will make temperature changes –Samples will be held in freezer until next session

Data Collection on 1-24 Agarose Gel Analysis of PCR Products –Pour agarose gels and load DNA samples –Apply electric current –Photograph resulting gel for data analysis Pouring the agarose Submerging the gel Loading the gel

Gel Electrophoresis Direction of Migration Larger fragments move more slowly; smaller fragments move more rapidly

Using the Micropipettor  Set to desired volume  Attach a clean tip  Push plunger to first stop  Insert tip into liquid  Release plunger SLOWLY  Position over well in gel  Push plunger SLOWLY to second stop to transfer solution or fill well  Discard tip in orange bag