08 January 2014

blast and RNA extraction

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Wednesday Jan 8

2014-01-08

ApGXL

RNA extractions for Phytotron samples. ‘Rz’ is RNAzol step, ‘Ry’ is RNeasy step, ‘conc’ is ng/ul and ‘qual’ is the 260/280 ratio.

Sample	Rz conc	Rz qual	Ry conc	Ry qual
04-A HS	23.06	1.69	17.12	1.54
04-B HS	12.89	1.81	11.27	1.42
05-B HS	63.73	1.66	14.48	1.82
06-A HS	80.00	1.69	11.19	1.46
06-B HS	31.24	1.70	5.68	1.15
08-A HS	47.97	1.65	8.32	1.55
09-A HS	22.68	1.60	5.31	1.11
10-A HS	42.40	1.59	9.11	1.35
10-B HS	13.10	1.61	15.18	1.34
12-A HS	38.57	1.69	14.38	1.52
12-D HS	21.5	1.57	14.85	1.33
13-A HS	17.44	1.63	9.24	1.24
13-B HS	10.58	1.49	9.34	1.30
16-A HS	6.31	1.57	9.76	1.45
16-B HS	15.26	1.59	5.55	1.00
21-A HS	19.00	1.64	8.97	1.29
21-B HS	16.85	1.58	4.83	1.21
22-A HS	13.55	1.38	5.73	1.19
22-B HS	22.9	1.67	11.35	1.35
22-C HS	66.41	1.66	21.77	1.62

Concentration and quality okay after RNAzol, but both decreased considerably after RNeasy. Disappointing.

Phone call with NuGen Tech Support

With: Ashesh About: Protocol for DGE and NGS library prep

Ovation DGE:

• input 10-100ng RNA
• okay to use MRC polyacryl carrier in RNA extraction
• normalize input RNA, at 100 ng RNA if possible, or max available for smallest concentration
• yields average 3 ug cDNA
• quantify DGE w/ Qubit
• purify with QIAquick PCR purification
• Bioanalyzer on a few samples after purification to check for problems before proceeding to library prep
• negative control not possible due to chimerica DNA/RNA amplification

Rapid DR multiplex NGS library prep:

• with SPIA-generated input cDNA use 500ng to 1 ug, preferably 1 ug input
• input cDNA needs to be fragmented!
• perform some controls with fragmented DNA - check Bioanalyzer traces
• 16 samples at a time. if less than 16, follow Low-plex Pooling Guidelines
• ethanol concentration important - make fresh daily
• run Kappa qPCR to determine library size