A-MAXD-2:maxdLoad2.bioinf.man.ac.uk:Wilson:[ImageAnalysisProtocol]EhV Microarray Image Analysis Protocol/0
Title: EhV Microarray Image Analysis Protocol. Description:
A-MAXD-2:maxdLoad2.bioinf.man.ac.uk:Wilson:[LabellingProtocol]EHV RT-PCR LABELLING/0
CY3/CY5 LABELLING RT-PCR REACTION All reactions perfromed in PCR machine (MJ Research PTC-200 Peltier Thermal Cycler). 25ug total RNA sample and 5ng spike mRNA (Stratagene mRNA spikes 1-10, 1ng/ul stock) in a total volume of 11ul, 2ug oligo-dT (0.5ug/ul) incubated at 70oC for 10 mins. Following snap cooling on ice, 12.6 ul of the reaction mix was added to each sample. Reaction mix composed of 5ul 5X first strand buffer, 3ul 0.1M DTT, 0.6ul 25mM dNTP mix (containing no dCTP) and 3ul Cy3- OR Cy5-dCTP (Amersham). Following the addition of 2ul of Superscript III Reverse Transcriptase (Invitrogen), samples were mixed gently and incubated at 42oC for 2 hours. Prior to hybridisation, samples were incubated at 70oC for 10 mins with 15ul 0.1M NaOH. 15ul of 0.1M HCl was added to neutralise the reaction.
A-MAXD-2:maxdLoad2.bioinf.man.ac.uk:Wilson:[ExtractionProtocol]EHV RNA EXTRACTION/0
RNA EXTRACTION PERFORMED USING RNA MIDIPREP KIT (QIAGEN). NOTE! 6 x 2ml RNALATER samples were processed using 6 midiprep columns the RNA eluted in 250ul and the samples pooled Extractions were performed in a PCR hood. 2ml Cellular samples were centrifuged (20,000g, 2 mins) and the pellet resuspended in 2ml RLT (+20 ul Beta mercapto ethanol). Following vigourous vortexing (1 min, in 5 second bursts), the samples were spun (20,000g, 5 mins) and the supernatant transferred to a 15 ml Falcon tube containing 2ml 70% ethanol. Following vigourous mixing the samples were applied to a Qiagen MidiPrep column, centrifuged (3,200g, 5 mins) and the flow-through discarded. Columns were washed twice with 2.5ml RPE buffer (3,200g, 5 mins) and transferred to a new Falcon tube. RNAse free water (250ul) was added, the samples incubated (room temperature, 1 min) and the RNA eluted by centrifugation (3,200g, 5 mins). Following the elution of RNA, replicate midipreps (6) were combined and the subsequent RNA solutions precipitated. 0.5 volumes of 7.5M NH4Ac and 2 volumes of 100% ethanol was added and the samples incubated at -80oC overnight. Following centrifugation (20,000g, 30 mins), the supernatant was discarded and the pellet washed twice with 0.5ml 70% ethanol (20,000g, 30 mins). The pellet was air dried, resuspended in 50 ul RNAse free water and stored at -80oC. RNA quantity and quality was assessed using the Agilent Bioanalyzer 2100 system (www.agilent.com)
Cultures were filtered through 0.45 um filters (Millipore) in 250 ml batches (i.e. 6 filters per 1.5 litre sample). The filtrate was discarded and the filters transferred to clean petri dishes. Cells from each filter were resuspended in 2ml of 1 x Phosphate buffered saline (PBS), centrifuged (20,000g, 5mins), resuspended (by vortexing) in 2ml RNAlater (Qiagen) and stored at -20oC until ready for processing.
A-MAXD-2:maxdLoad2.bioinf.man.ac.uk:Wilson:[TreatmentProtocol]Infect with EhV86/3
Cultures (1.5 litres) were infected with 3ml of EhV86 viral lysate (i.e. 2ml per 1litre) Viral lysate produced previously by filtering (0.45 um, millipore) a recently lysed culture of Emiliania huxleyi CCMP1516 infected with EhV86.
A-MAXD-2:maxdLoad2.bioinf.man.ac.uk:Wilson:[TreatmentProtocol]Infect with nothing/1
Cultures (1.5 litres) were 'infected' with 3ml of fresh f/2 medium (i.e. 2ml per 1 litre).
A-MAXD-2:maxdLoad2.bioinf.man.ac.uk:Wilson:[TreatmentProtocol]Splitting of culture/0
10 litre culture was split into 6 x 1.5 litre cultures (2 litre Duran bottles) when the cell number reached 1x10~6 cells per ml. The remaining 1 litre was split into 2 x 500ml cultures, for control cultures (uninfected and infected control). Infected control cultures lysed (typically) 4 days post infection.
A-MAXD-2:maxdLoad2.bioinf.man.ac.uk:Wilson:[SamplingProtocol]EHV Growth conditions/0
E. huxleyi cultured in f/2 media (supplemented with nutrients) on a 16:8 hour (light:dark) cycle at 15oC. Dark cycle begins at 04:30 and ends at 12:30. 20ml of exponentially growing E. huxleyi cells (1x10~6 cells/ml) innoculated per 1 litre of fresh media. f/2 Medium (Guillard & Ryther 1962, Guillard 1975) Guillard, R.R.L. 1975. Culture of phytoplankton for feeding marine invertebrates. pp 26-60. In Smith, W.L. and Chanle,y M.H. (eds.) Culture of Marine Invertebrate Animals. Plenum Press, New York, USA. Guillard, R.R.L. and Ryther, J.H. 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8: 229-239.