User:Mark X. Ling/Notebook/Micb 323/2009/03/03

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Part 1: Souther Blot
A. Probe hybridization Steps completed by instructor: 1)2x SSC ==>move to the bottle after rolling==>discard 2)20mL PREhybridization buffer ==>oven for 1hr==>discard 3)20mL Hybridization buffer[w/ probe] ==> overnight Washes: 4 washes(2x 2 types of washes) 1)20mL of wash buffer "#1" to bottle==>oven for 20mins==>discard 2)repeat 1) 3)20mL of wash buffer "#2" to bottle after 2) ==>oven for 20mins ==> discard 4) repeat 3)
 * probe used: PCR product with DIG-dUTP incorporated.

B.Detection: DIG buffer 1(1"){5min}>DIG buffer 2 [blk](10mL){30min}> DIG antibody (10mL){30mins} > DIG buffer 1 {15min} > DIG buffer 1{15min}>DIG buffer 3(10mL){2-5min}>CSPD(5mL){5min} >let excess off[not completely dry]>X-ray cassette >[dark room]Expose (10-20min){record time}

Part 2:Macrophage Preparation
Room 109 ==>10ng/mL of LPS and IFNgamma
 * was student A
 * I stimualted LPS and IFNg samples
 * some of the media collected has PBS in it
 * media is more diluted


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