2/15/18

Today we finished the Ludox protocol and also performed the E.Z.N.A. Tissue DNA Protocol

Procedure: 

Ludox Cell Layer

1. Spin 5 minutes at 3000 g
2. Remove supernatant
3. Add 1 mL of PBS to wash the cells
4. Spin for 5 minutes at 3000 g
5. Remove supernatant
6. Resuspend cells in 100 uL of PBS and combine to one tube
7. Add 20 uL of Iodine and 20 uL of cells
8. Observe under microscope with 5 drops of 2 uL

Before Starting

1. Prepare DNA Wash Buffer and HBC Buffer according to direction in the protocol guide
2. Heat Elution Buffer to 70 C in heat block
3. Chill PBS to 4 C

Prepare Cell Suspension

1. Add 200 uL of PBS and flick tube to mix.  Remove the supernatant without getting rid of the pellet
2. Add another 200 uL of PBS
3. Add 25 uL of OB Protease then vortex

Lysis

1. Add 220 uL of BL Buffer
2. Incubate at 70 C for 10 minutes in heat block then vortex

Binding

1. Add 220 uL of 100% ethanol then vortex
2. Transfer all of the sample to the sample into an HiBind with a 2 mL Collection Tube
3. Centrifuge at max speed for 1 minute
4. Discard the filtrate

Wash and Dry

1. Add 500 uL of HBC Buffer to HiBind
2. Centrifuge at max speed for 30 seconds
3. Discard filtrate and collection tube
4. Obtain a new collection tube for the HiBind
5. Add 700 uL of DNA wash buffer
6. Centrifuge at ma speed for 30 seconds
7. Discard filtrate
8. Repeat steps 5-6
9. Centrifuge empty HiBind on max speed for 2 minutes

Elute

1. Transfer the HiBind into a nuclease free 1.5 mL microcentrifuge tube with a name and date
2. Add 100 uL Elution Buffer heated to 70 C
3. Let sit at room temperature for 2 minutes
4. Centrifuge at max speed for 1 minutes  and discard the HiBind and keep the liquid in the tube
5. Store at -20 C

Observation

There were 14, 17, and 25 cells in each drop from one slide and 22, 25, 32 cells in each drop from the other slide.

Thoughts

We accidentally added Iodine to our cell solution which messed up our color.  We will see how that changes our data.  We labeled our tube as Sec 22, Group 8, 2-15-18, DNA on the top of the tube with KSA on the side of the tube