To catch everyone up, we got the new kit on Monday last week. And we put it to use right away.
30 mL of ethanol was added to wash buffers 1 and 2
box on the bottle was checked to show that we had already added the ethanol on both bottles
350 mili liters of lysis buffer A was pipetted into a 1.5 mL microcentrifuge tube
About .95 g or 95 mg of fresh plant tissue Was measured out
The material was ground with a pencil with a cap into a microcentrifuge tube
The tissue powder was transferred into a 1.5 mL microcentrifuge tube that contained 350 milliliters of lysis buffer A and then vortexed for 15 seconds
50 mili liters of lysis buffer B and 29 mili liters of RNase A were added
the sample was put in a DryBlockHeater for 10 minutes at 65 degrees Celsius. The vortex was used once every minute for 15 seconds during the dry block heating
130 milliliters of precipitation solution was added and The tube was inverted twice
The microcentrifuge tube was put in an ice bath for more than 5 minutes
The sample was centrifuged at 12500 for 15 minutes
The supernate was collected and then put into a new microcentrifuge tube
The next day 400 milliliters of plant gDNA Binding solution and 400 milliliters of 96 percent ethanol were added to the supernate and mixed
Six hundred milliliters of the prepared mixture were transferred to the spin column and centrifuged at 6,000 x g for 1 minute. The flow-through solution was discarded. The remaining mixture was applied to the same column and centrifuged at 6,000 x g for 1 minute
Five hundred milliliters of wash buffer one was added to the column and centrifuged at 12,500 for 5 minutes. The collection tube was emptied, the purification column was put back into the tub, and then re-spun at 12,500 for 5 minutes.
The collection tube containing the flow-through column was discarded and transferred to a sterile 1.5 mL microcentrifuge tube.
One hundred milliliters of elution buffer was added to the center of the column membrane. It was incubated at room temperature for 5 minutes and then centrifuged at 8,000 x g for 1 minute.
A second elution step was performed using 100 milliliters of elution buffer in the same tube.
The microcentrifuge tube was put in a freezer at -20 degrees celsius until the next day when we put 2 ml into the machine that sent information to the computer on how pure the DNA is. Here is a picture of the results
