PP7 Tethering assay

'''PP7 Tethering Assay '''

''' Growing cultures: '''

Day1: Start 5ml overnight culture in synthetic selection media containing 2% dextrose media

Day 2:


 * Dilute starter culture to O.D.600 0.1 in 20ml media.
 * Grown to mid log phase (O.D.600 0.4-0.7) at 30°C.
 * Collect ~15ml cells by centrifugation (spin at 3000rpm, wash 1X in PBS) and freeze in liquid nitrogen.

''' Extract preparation: '''
 * Resuspend yeast pellet in 800ul 1X PBST (with RNAsin and protease inhibitors).
 * Lyse cells with 2x30second pulses with the bead-beater.
 * Clarify extract by centrifugation (10min at 13,000rpm at 4°C ).
 * Measure protein concentration by Bradford Assay and normalized protein concentration of all samples.
 * Prepare protein samples by resuspending 50ul extract in 50ul 2X SDS sample buffer.
 * Boil protein samples for 2 min.

''' RNA isolation: '''
 * RNA isolation is done using the Qiagen RNeasy RNA isolation kit (modified protocol).
 * Combine 100ul extract, 200ul buffer RLT and 300ul 70% EtOH.
 * Add to column, centrifuge 15sec at 10,000rpm.
 * Wash with 500ul RPE, spin 15 sec at 10,000rpm.
 * Wash with 500ul RPE, spin 2 min at 10,000rpm.
 * Dry column with 1 min spin @ full speed.
 * Elute RNA in 50ul RNase free dH2O.

''' DNase digestion of RNA samples: '''
 * Add 5ul 10X DNase to each sample and 1-2ul DNase (Ambion RNase-free DNase).
 * Incubate for 30 min at 37°C.
 * Stop reaction with 4ul DNase inactivating beads.
 * Spin for 3min at full speed.
 * Transfer sup to new tube and spec samples with nanodrop.