User:Eric M. Walters/Notebook/Spring 2012/2012/06/11

Cyanobacterial acrylate production

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acsA::barcode - the story so far: 5/21 - Amplified the BC-front & BC-back regions with v2 & BC primers 5/22 - gel verified 5/22 - 45:00 digest + heat inactivated 30:00 50 uL ligation (gel verified) Amplified w/ v2 UpF/DnR primers 5/23 - gel verified 5/23 - Transformed OD=.96 culture w/ 3.2mg (Alongside wt 7002 control) 5/24 - Plated transformations on 500 uM AA 5/29 - 18:9::ligation:control Colony PCR w/ v1 UpF/DnR 5/30 - No positives, but could have been swamped by primers amplifying wt acsA as well 5/30 - BC-F+DnRv1 colony PCR off of transformant plate: all hits ~550bp Though v1 primers (v2 used for construct), overlap means they could still amplify from end 5/31 - Made 5 mM AA plates to enrich, plated from transformation onto it 6/4 - Colony PCR w/ BC-F+DnRv1 off non- transformation plate. Gel annotat- ion is incorrect since "flanking" v1 primers still hella overlap No hits whatsoever 6/6 - Colony PCR off 10 mM AA plates with BC-F & DnRv1 6/7 - Gel of 6/6. Faint ~200bp bands from all colonies, not in transformation construct, which had large 500bp I expected, along with some other larger ones (?). The small bands in the colonies were not present in the gDNA control though, so what it amplified must be somehow related to the acrylate tolerance. 6/8 - Flanking (UpFv1+DnRv1) cPCR off 10 mM patches. Patches 1, 5, 9, 12 had 3 kb bands indicative of acsA+up+dn but gDNA didn't?? Also ~500bp bands in 1, 2, 3, 6, 8, 9, 10, 12, gDNA Stop... rewind! Looks like I didn't actually have any positive hits on the acsA::BC cloning, so I'm going to transform again and plate on higher (5 mM) AA from the get go. 2x50 uL amplifications a la 5/22 Per 50 μL: 35 μL H2O 10 μL Buffer HF 1 μL dNTPs 1 μL acsA-upFv2 1 μL acsA-dnRv2 1 μL heat-inactivated ligation product 1 μL Phusion Given 200 nM primer concentration and Phusion, the annealing temp is 71°C Given the construct is a bit over 1 kb, giving it 1:10 extension time Run overnight.