4 30 2015 RNA Isolation FISH 310 samples

Today I isolated RNA from samples collected by the Biology of Shellfish students. These samples are nearly identical to mine except that they were treated slightly different and collected by the students. The tissue is primarily ctenidia but there were traces of mantle and glycogen stores in it. The samples were collected on tuesday and stored in RNAzol in the -80 until today.

The samples were labeled
42815HM1-5
42815SM1-5

The protocol is as follows:

1. 100 mg tissue homogenized in 1 ml RNAzol RT at room temp. 
2. Added 400 ul Nanopure water to homogenate.
3. Incubated 15 minutes at room temp
4. Centrifuged 15 minutes at 11,400 rpm (~12,000 g)
5. Transfered 1 ml supernatant to fresh tube
6. Added 400 ul 75% EtOH
7. Mixed via inversion for 15 seconds
8. Incubated samples 10 minutes at room temp
9. Centrifuged 8 minutes at 11,400 rpm (~12,000 g)
10. Discarded supernant
11. Added 600 ul 75% EtOH
12. Vortexed 15 seconds
13. Centrifuged 3 minutes at 7,400 rpm (~8000 g)
14. Repeated steps 10 - 13. 
15. Removed supernatant
16. resuspended RNA in 100 ul Nanopure water
17. Nanodropped

The results from the nanodrop were as follows. 

Sample ID	Date	ng/ul	A260	A280	260/280	260/230
42815HM1	4/30/2015	94.49	2.362	1.336	1.77	2.25
42815HM2	4/30/2015	46.68	1.167	0.741	1.57	0.88
42815HM3	4/30/2015	40.23	1.006	0.649	1.55	0.67
42815HM4	4/30/2015	105.78	2.644	1.475	1.79	1.43
42815HM5	4/30/2015	182.93	4.573	2.517	1.82	2.19
42815SM1	4/30/2015	257.87	6.447	3.468	1.86	2.29
42815SM2	4/30/2015	66.35	1.659	0.999	1.66	2.26
42815SM3	4/30/2015	43.2	1.08	0.645	1.67	0.99
42815SM4	4/30/2015	143	3.575	2.001	1.79	1.24
42815SM5	4/30/2015	235.27	5.882	3.195	1.84	1.69

Tomorrow I will run a qPCR on the RNA to verify the purity of the RNA and ensure there is no genomic DNA in the sample.