User:Howard Boland/Notebook/Art from Synthetic Biology/2010/10/25

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Gel PCR Products(pUA66katE template, 2359bp) After revising optimised condition. I ran the new PCR products on a 2% Gel - this should have been run on a 1% Gel. Lane 1: 1kb NEB Quick Ladder Lane 2: BLANK Lane 3: Undigested PCR Product, expected 2359bp (from pUA66katE - hereby named pSense66) Lane 4: BLANK Lane 5: Undigested PCR Product, expected 2359bp (from pUA66katE - hereby named pSense66) Lane 6: BLANK Lane 7: 5µL pMAK512 (template for the pBR322 pcr) Lane 8: 5µL pUA66katE (template for this pcr) Error: PCR product is not completely optimised Gel PCR Products(pMAK512 template, 790bp, pBR322) I ran the PCR products from the pMAK512 template on a 2% Gel - this should have been run on a 1% Gel. Lane 1: 1kb NEB Quick Ladder Lane 2: BLANK Lane 3: Undigested PCR Product, expected 790bp (from pMAK512 - hereby named pBR322) Lane 4: BLANK Lane 5: Undigested PCR Product, expected 790bp (from pMAK512 - hereby named pBR322) Lane 6-8: BLANK Error: Should have been run on a 2% Gel PCR Product PstI Digestion Master Mix: 10µl 10xNEB#3 1µl 100xBSA 3µl PstI 26µl H2O I used one tube of each of the PCR reactions (pSense66 and pBR322) to complete the reactions I set up the following Tube 1: Add 20µl Master mix to 30µl pBR322 PCR product Tube 2: Add 20µl Master mix to 30µl pSense66 PCR product Digest for 2 hours at 37ºC Gel PCR Products, Digest and Undigested Lane 1: 1kb NEB Quick Ladder Lane 2: Digested PCR Product with PstI, expected 2359bp (from pUA66katE - hereby named pSense66) Lane 3: BLANK Lane 4: Digested PCR Product with PstI, expected 790bp (from pMAK512 - hereby named pBR322) Lane 5: BLANK Lane 6: Undigested PCR Product, expected 2359bp (from pUA66katE - hereby named pSense66) Lane 7: BLANK Lane 8: Undigested PCR Product, expected 790bp (from pMAK512 - hereby named pBR322) Overnight PCR Product PstI Digestion Master Mix: 10µl 10xNEB#3 1µl 100xBSA 3µl PstI 16µl H2O I used one tube of each of the PCR reactions (pSense66 and pBR322) to complete the reactions I set up the following Tube 1: Add 15µl Master mix to 25µl pBR322 PCR product Tube 2: Add 15µl Master mix to 25µl pSense66 PCR product Digest overnight at 37ºC