HaeIII and BstEII restriction enzymes that cut at specificsites in the amplified sequence of interest were used. All tubeswere kept on ice until samples were ready to incubate samples inthe Thermocycler.

Tubes #1, #2, #3 were placed at 37C for two hours. Tubes #4, #5,#6 were placed at 60C for two hours.

Make a prediction of which enzyme will cut WT vs. shy2-2vs F2. ? Question: why must the tubes be put into differenttemperatures?

For plant physiology

HaeIII BstEII Tube # Contains: Add: Add: Add: GGCC CCGG GGTNACC CCANTGG 1 0.2ul Haelll 7.8ul H20 2.Oul Buffer 10ul PCR Product #1: WT 2 0.2ul Hae!!! 7.8ul H20 2.Oul Buffer 10ul PCR Product #3: shy2 3 0.2ul Hae!!! 7.8ul H20 2.Oul Buffer 10ul PCR Product #5: F2 4 0.2ul BstEll 7.8ul H20 2.Oul Buffer 10ul PCR Product #1: WT 5 0.2ul BstEll 7.8ul H20 2.Oul Buffer 10ul PCR Product #3: shy2 6 0.2ul BstEll 7.8ul H20 2.0ul Buffer 10ul PCR Product #5: F2