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. 

3/1/9- RNA QUANTIFYING

  • 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
136116
112192
100Too high
124Too high
11598.2
106110
126Too high
140102