diff options
| author | Bonface | 2024-02-15 06:09:54 -0600 |
|---|---|---|
| committer | Munyoki Kilyungi | 2024-08-09 13:30:43 +0300 |
| commit | e34e7da50fc0ff5ed41e8bdaf2b1d41c9e9cf534 (patch) | |
| tree | 67c6bdeb413af7d1dd6c4d02f37b206850a78531 /general/datasets/B30_K_1206_M/experiment-design.rtf | |
| parent | b2feda451ccfbeaed02dce9088d6dd228cf15861 (diff) | |
| download | gn-docs-e34e7da50fc0ff5ed41e8bdaf2b1d41c9e9cf534.tar.gz | |
Update dataset RTF Files.
Diffstat (limited to 'general/datasets/B30_K_1206_M/experiment-design.rtf')
| -rw-r--r-- | general/datasets/B30_K_1206_M/experiment-design.rtf | 62 |
1 files changed, 0 insertions, 62 deletions
diff --git a/general/datasets/B30_K_1206_M/experiment-design.rtf b/general/datasets/B30_K_1206_M/experiment-design.rtf deleted file mode 100644 index e743086..0000000 --- a/general/datasets/B30_K_1206_M/experiment-design.rtf +++ /dev/null @@ -1,62 +0,0 @@ -<blockquote> -<p><strong>RNA Sample Processing:</strong></p> - -<p>Trizol RNA isolation and RNeasy clean up protocol for whole plants (embryo-derived tissue dissected from 4 days old germinating grains) and the seedling leaves (12 days after planting).</p> - -<p><br /> -☠Grind tissue (9 embryos) with a mortar and pestle in liquid nitrogen<br /> -☠Add 5 ml TRIzol (pre-heated to 60oC) to all samples, vortex until all the tissue is thawed, place in the 60oC waterbath..<br /> -☠Incubate samples at 60oC for 10 minutes, vortexing three times.<br /> -☠Centrifuge @ 4000 x rpm @ 4C for 30 minutes (in Eppendorf 5810R).<br /> -☠While centrifuging, label new set of 15 ml tubes<br /> -☠Transfer supernatant to 15 ml centrifuge tube<br /> -☠Add 1 ml of chloroform. Vortex the sample until color shade is uniform at least 5<br /> -seconds, and incubate at room temperature for 5 minutes.<br /> -☠Centrifuge @ 4000 x rpm for 30 minutes @ 4oC.<br /> -☠While centrifuging, label new 15 ml tubes<br /> -☠Collect the upper aqueous layer (there will be about 3 mls) and transfer to a new 15 ml tube.<br /> -☠Add 0.6 volumes (2 ml) of isopropanol, mix gently, incubate at room temperature for 20 minutes.<br /> -☠Centrifuge @ 4000 rpm for 30 minutes @ 4oC.<br /> -☠Wash the pellet with 10 ml of cold 75% ethanol. Swirl & centrifuge at<br /> -4000 rpm for 15 minutes @ 4oC.<br /> -☠Discard supernatant, centrifuge for 5 min, remove the rest of the ethanol<br /> -☠Air-dry the pellet for 10 minutes, inverted on a kimwipe.<br /> -☠Dissolve pellet in 400 ul of DEPC-treated H2O. Resuspend by pipeting up & down a<br /> -few times.<br /> -☠Add 2 ul SuperaseIn. Incubate at 60oC for 10 minutes to resuspend.<br /> -☠Set water bath to 37oC.<br /> -☠Add 50 ul 10X DnaseI Buffer, 45 ul H2O and 5 ul of DnaseI, incubate at 37oC for 1 hr.<br /> -☠Prepare Buffer RLT (Rneasy Clean-up Midi Kit) by adding b-mercaptoethanol (10ul/1ml RLT).<br /> -☠Add 2.0 ml Buffer RLT to the RNA prep and mix thoroughly.<br /> -☠Add 1.4 ml ethanol (96-100%) to the diluted RNA. Mix thoroughly.<br /> -☠Label 15 ml tubes from the kit and place midi columns in them<br /> -☠Apply sample to a Midi column, close tube gently and centrifuge for 20 min at 3000 rpm.<br /> -☠Discard the flow-through.<br /> -☠Add 2.5 ml Buffer RPE to the RNA easy column, close the centrifuge tube gently,<br /> -incubate for 3 min<br /> -☠Centrifuge for 10 min at 3000 rpm. Discard the flow-through.<br /> -☠Add another 2.5 ml Buffer RPE to the RNeasy column. Close the centrifuge tube<br /> -gently, incubate for 3 min<br /> -☠Centrifuge for 10 min at 3000 rpm, remove flow-through<br /> -☠Centrifuge again for another 5 min.<br /> -☠Label new 15 ml tubes from the kit.<br /> -☠Transfer the RNA easy column to a new tube and pipet 250 ul volume of<br /> -RNase-free water directly onto the RNeasy silica-membrane incubate for 1 min<br /> -☠Centrifuge for 5 min at 3000 rpm.<br /> -☠To the same tube add again 250 ul H2O, incubate for 1 min.<br /> -☠Centrifuge for 5 min at 3000 rpm.<br /> -☠Label two sets of 1.5 ml Eppendorf tubes.<br /> -☠Transfer 490 ul to the one tube and 10 ul to another one. Use 10 ul tube for the RNA</p> - -<p>Detailed descriptions of these procedures can be found under the ArrayExpress (http://www.ebi.ac.uk/aerep/?) protocol P-MEXP-4631 (Caldo et al. 2004).</p> - -<p><strong>Replication and Sample Balance:</strong></p> - -<p>3 independent replicates of both parental cultivars Steptoe and Morex were generated for both tissues, embryo and seedling leaf.</p> - -<p><strong>Experimental Design and Batch Structure:</strong></p> -</blockquote> - -<blockquote> -<p>The following are ArrayExpress (http://www.ebi.ac.uk/aerep/?) experiment IDs: E-TABM-111 (leaf, 41 chips) and E-TABM-112 (embryo derived, 156 chips).</p> -</blockquote> |