Homogenization and RNA treatment: round 2 of samples

3/4/19- 9:40 AM

Using samples 101, 103, 110, 116, 122, 128, 138, 141

I removed the eight samples from the -80 and immediately put them on dry ice. Once I began homogenizing the tissue using the pestle and mortar, I had some trouble getting all of the tissue homogenized for the first two samples (138, 122), so I did not keep half of the powder. 122 and 141 also fell out of the bowl of liquid N2 during grinding, so we’ll see what turns up with that. 116 was a very small sample, so I did not end up getting much powder into the buffer. This time, I did step 3 (DNA spin column) and kept the columns for 128, 138, 110, and 101.

Put everything (original sample tubes with half of powder, current samples with completed buffer, and DNA columns) into Box 3 in the -80. Will return to do treatment this week. 

3/7/19- 8:30 AM

I took the homogenized samples out of the -80 and began the treatment yo extract RNA. Spin columns clogged again, so I increased the speed to last week’s (full speed). I kept the spin columns from the last step (10), but did not keep the flow through.

Everything is in the -80 this time, in 2 boxes labeled with my name, “samples,” and date, under Laura’s samples. Next week I will quantify these and begin the third round of samples.

RNA extraction for first round of oyster tissue samples

Alanna Greene

See Laura’s notebook entry for homogenization steps.

Realized we skipped step 3 (centrifuging again in a DNA spin column to remove DNA). We will see if we can salvage RNA, if not we will re-d0 this with different samples. 

2/22/19 1:30 p.m. 

Samples were removed from -80 and I began the treatment on the samples, starting at step 3 in the QIAGEN RNeasy Plus protocol. Sample 136 clogged the MiniElute column (part of homogenate did not flow through after centrifuging ). I transferred this to a new column called 136B, which I processed over to replace 136, which was completely clogged. It turns out a lot of the columns were clogged (step 6+7), so at step 7 I increased the speed to 16G in the centrifuge. Once the protocol was completed, I stored the samples in the fridge. 


  • Autoclaved dishes and got more liquid nitrogen 
  • Ran samples through Qubit 30 following protocol: 
    • Wiped down tubes 
    • Calibrated standards first (blank and full) 
  • For samples: 1 microliter in sample in 199 microliter in solution
  • Successfully quantified! 
Sample # Qubit Reading 
S1Too low out of range 
S29.92 ng/mL
100Too high
124Too high
126Too high