User:Joytafty/Notebook/Effects of EphA2 and CXCR3 KD in hMVECs/2012/04/09: Difference between revisions
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'''Materials:''' | '''Materials:''' | ||
26g needles and syringes | * 26g needles and syringes | ||
RNAse free Eppendorb tubes | * RNAse free Eppendorb tubes | ||
RNAse free pipette tips | * RNAse free pipette tips | ||
Chloroform (Sigma, Calbiochem); | * Chloroform (Sigma, Calbiochem); | ||
RNEasy Mini Kit (Qiagen 74104 (50) or 74106 (250); | * RNEasy Mini Kit (Qiagen 74104 (50) or 74106 (250); | ||
DNAse/RNAse free water (MP Biomedicals 821739); | * DNAse/RNAse free water (MP Biomedicals 821739); | ||
DEPC-treated water (Ambion AM9922, AM9915G); | * DEPC-treated water (Ambion AM9922, AM9915G); | ||
RNase/DNase free tubes (VWR 87003-294); | * RNase/DNase free tubes (VWR 87003-294); | ||
70 % Ethanol/RNAse free water | * 70 % Ethanol/RNAse free water | ||
'''Procedure:''' | '''Procedure:''' | ||
Preparation | [[Preparation]] | ||
Spray down everything with RNaseAway water | * Spray down everything with RNaseAway water | ||
Thaw sample on ice | * Thaw sample on ice | ||
Homogenize sample | [[Homogenize sample]] | ||
Pass sample through 26g needle and syringe 12 - 15 times | * Pass sample through 26g needle and syringe 12 - 15 times | ||
Vortex to mix 5 - 10 s | * Vortex to mix 5 - 10 s | ||
Separate protein and DNA | [[Separate protein and DNA]] | ||
Add 100 µL chloroform | * Add 100 µL chloroform | ||
Invert sample a few times by hand, then vortex 5 s | * Invert sample a few times by hand, then vortex 5 s | ||
Let sit at RT 2-3 mins to allow sample to settle into 2 layers | * Let sit at RT 2-3 mins to allow sample to settle into 2 layers | ||
COLD ROOM: Centrifuge 15 mins at 12000 RPM at 4C | * COLD ROOM: Centrifuge 15 mins at 12000 RPM at 4C | ||
warm DEPC water to 65 C, label Eppendorf collection tubes | * warm DEPC water to 65 C, label Eppendorf collection tubes | ||
Take sample from cold room centrifuge. | * Take sample from cold room centrifuge. | ||
Carefully remove top layer of sample into labeled collection tubes | * Carefully remove top layer of sample into labeled collection tubes | ||
Keep track of sample volume (90 - 95 µL) | * Keep track of sample volume (90 - 95 µL) | ||
Put the bottom layer to -80C | * Put the bottom layer to -80C | ||
KEEP ON ICE FROM THIS POINT ON | KEEP ON ICE FROM THIS POINT ON | ||
Add 150 µL 70% EtOH/RNaseAway water to sample. Mix by tipping/pipetting. | [[Collect RNA onto column]] | ||
Remove sample and place in RNEasy column with collection tube. | * Add 150 µL 70% EtOH/RNaseAway water to sample. Mix by tipping/pipetting. | ||
Prepare RNAse free tubes to hold columns. | * Remove sample and place in RNEasy column with collection tube. | ||
Centrifuge sample 15 s at > 10000 RPM | * Prepare RNAse free tubes to hold columns. | ||
Return eluent to top of filter column | * Centrifuge sample 15 s at > 10000 RPM | ||
Repeat spin for 15 s at > 10000 RPM | * Return eluent to top of filter column | ||
Repeat a - c if sample remains | * Repeat spin for 15 s at > 10000 RPM | ||
Buffer RW1 Rinse | * Repeat a - c if sample remains | ||
Add 700 µL Buffer RW1 to columns | [[Buffer RW1 Rinse]] | ||
Centrifuge 15 s at > 10000 RPM | * Add 700 µL Buffer RW1 to columns | ||
Discard flowthrough | * Centrifuge 15 s at > 10000 RPM | ||
* Discard flowthrough | |||
Transfer sample to new collection tube | [[RPE washes]] | ||
Add 500 µL RPE wash buffer to columns | * Transfer sample to new collection tube | ||
Centrifuge 15 s at > 10000 RPM | * Add 500 µL RPE wash buffer to columns | ||
discard flowthrough | * Centrifuge 15 s at > 10000 RPM | ||
Add another 500 µL RPE wash buffer | * discard flowthrough | ||
Centrifuge 2 min at > 10000 RPM | * Add another 500 µL RPE wash buffer | ||
Transfer sample to new collection tubes | * Centrifuge 2 min at > 10000 RPM | ||
Centrifuge 1 min at > 10000 RPM to remove all ethanol | * Transfer sample to new collection tubes | ||
RNA elution | * Centrifuge 1 min at > 10000 RPM to remove all ethanol | ||
Add 20 µL DEPC water (heated to 65 C) to filter | [[RNA elution]] | ||
Incubate filter at RT 2 - 3 mins | * Add 20 µL DEPC water (heated to 65 C) to filter | ||
Centrifuge for 2 mins at > 10000 RPM in clean Eppendorf tubes to elute. | * Incubate filter at RT 2 - 3 mins | ||
RNA purity check | * Centrifuge for 2 mins at > 10000 RPM in clean Eppendorf tubes to elute. | ||
Wipe everything down with RNAseAway water | [[RNA purity check]] | ||
Bring samples on ice to Nanodrop | * Wipe everything down with RNAseAway water | ||
* Bring samples on ice to Nanodrop | |||
Run samples on Nanodrop | Run samples on Nanodrop | ||
Revision as of 05:54, 9 April 2012
 Project name
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<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page |
RNA extraction of 2012/03/30 samplesGoal:
Background:
Materials:
Procedure: Preparation
KEEP ON ICE FROM THIS POINT ON Collect RNA onto column
Run samples on Nanodrop How to run Nanodrop: Open the Nanodrop Application. Select the “Nucleic Acid” tab. a. Before being able to use the program, the Nanodrop needs to be initialized. Clean the sample pedestal RNAse free water and a Kimwipe. Add 2 uL of RNAse free water, put top arm down and click OK. Make sure that the liquid between the node surface of the top arm gets into contact with the pedestal. It should have an hourglass like appearance. b. Select RNA-40 in the sample type window in the upper right corner. c. Wipe off sample pedestal and blank the device with 2 uL of buffer that sample is in (from this protocol, it is DEPC-treated water). d. Wipe off the pedestal and pipette 2 uL of the sample on to measure. Note: Read only a volume of sample once. If make an additional read on the sample, it will change. To get replicate readings, load another 2 uL of the sample on.
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