Experiment 6: Southern Blot & Macrophage Preparation
Part 1: Souther Blot
A. Probe hybridization
- probe used: PCR product with DIG-dUTP incorporated.
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)
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
- I stimualted LPS and IFNg samples
- some of the media collected has PBS in it
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