User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/12

Project name

Main project page Previous entry      Next entry

Agarose Gel Extraction Gels were prepared using 0.6g of Agarose powder with 60ml of water, heated until no specks remained and then cooled. 5µl of Ethidium Bromide was added to the gel. Gel: to extract a doubled digested vector based on the digestion User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/11#Double digestion pUA66 XhoI / BamHI Lane 1: 10µl 1kb Quick NEB Ladder Lane 2: 25µl Digest pUA66#A, XhoI/BamHI Lane 3: 25µl Digest pUA66#A, XhoI/BamHI Lane 4: Blank Lane 5: 5µl Digest pUA66#B, XhoI/BamHI Lane 6: 5µl Digest pUA66#B, XhoI/BamHI Lane 7: Blank Lane 8: Blank Gel Result: Gel Purification The gel bands were cut and weighted with the following results: pUA66#A1: 130mg pUA66#A2: 117mg pUA66#B1: 162mg pUA66#B2: 157mg From this the following measurements where used: Add 1:3 volume of QG Buffer pUA66#A1, 130x3 = 390µl QG Buffer pUA66#A2, 117x3 = 351µl QG Buffer pUA66#B1, 162x3 = 486µl QG Buffer pUA66#B2, 157x3 = 471µl QG Buffer Mix well, place in 47ºC water bath for 10 minutes, mixing ever 2-3 minute to dissolve gel Add 1:1 volume of Isopropanol pUA66#A1, 130µl Isopropanol pUA66#A2, 117µl Isopropanol pUA66#B1, 162µl Isopropanol pUA66#B2, 157µl Isopropanol For each tube place apply to separate column Centrifuge for 1 minute at 13,000 rpm and discard flow-through For each column add 500µl QG Buffer Centrifuge for 1 minute at 13,000 rpm and discard flow-through Add 750µl of PE wash with Ethanol Centrifuge for 1 minute at 13,000 rpm and discard flow-through Centrifuge for an additional minute at 13,000 rpm and discard flow-through Place each column in a separate Eppendorf tube For each column add 30µl water and wait for 1 minute To elute centrifuge for 1 minute at 13,000 rpm, label and keep tube stored at -20ºC until use. Gel Purification Check Quality assurance before use in transformation. Lane 1: 5µl 1kb Quick NEB Ladder Lane 2: 2µl Digest pUA66#A1, XhoI/BamHI/Purified Lane 3: 2µl Digest pUA66#A2, XhoI/BamHI/Purified Lane 4: Blank Lane 5: 2µl Digest pUA66#B1, XhoI/BamHI/Purified Lane 6: 2µl Digest pUA66#B2, XhoI/BamHI/Purified Lane 7: Blank Lane 8: Blank Gel Result: Nanodrop Sample ng/µl 260/280 pUA66#1-11082010-X-B-Purified 5.31 2.01 pUA66#2-11082010-X-B-Purified 6.46 1.86 pUA66#3-11082010-X-B-Purified 15.01 0.98 pUA66#4-11082010-X-B-Purified 8.92 1.81 pUA66#5-11082010-X-B-Purified 12.52 2.11 pUA66#6-11082010-X-B-Purified 9.13 1.92 pUA66#A1-12082010-X-B-Purified 22.63 1.69 pUA66#A2-12082010-X-B-Purified 19.44 1.62 pUA66#B1-12082010-X-B-Purified 8.01 1.80 pUA66#B2-12082010-X-B-Purified 22.55 1.89 Ligation 4 Ligations were setup 1µl 10xLigation Buffer NEB 1µl T4 Ligation NEB 1µl of Template DNA pUA66#A1, 1µl pUA66#A1, 1µl pUA66#B2, 1µl pUA66#B2, 1µl For each tube add 8µl katE promoter Incubate at room temperature for 2 hours Transformation Six transformation were setup Transfer 6 tubes of competent cell straight onto ice for 5 minutes For each tube add DNA template pUA66#A1 10 µl pUA66#A2 10 µl pUA66#B1 10 µl pUA66#B2 10 µl pUA66#A1 (8µl water, 2µl pUA66#A1), without T4 pUA66#4 Undigested, 1µl Wait for an additional 5 minutes Place in 42ºC water bath for 1 minute Place back on ice for 5 minutes Add 250µl SOC (should be 37ºC) Incubate in shaker for 1 hour Add the entire content of each onto a Kanamycin LB-Agar plate and use glass beads to spread Incubate overnight PCR, katE Generate insert using colony PCR on 4 tubes each of 50µl volume Colony Dillution Add 10µl water to an Eppendorf tube Add one colony E.coli Mix well Master mix (4x) 100µl Water 20µl 10xOptimized DyNAzyme Ext Buffer 4µl 10mM dNTP 8µl Primer: HB katE F 8µl Primer: HB katE R Reaction Aliquot 35µl Master Mix into each of four 0.5ml PCR tube Add 1µl Template DNA from the Colony Dilution For each tube add 5µl of DyNAzyme EXT DNA Polymerase (possible error - as I might not have added this) Place in PCR thermocycler Reaction Environment Lid: 100ºC; Volume: 40µl 94ºC for 05:00 94ºC for 00:30 55ºC for 00:50 72ºC for 10:00 GOTO step 2, 32 times 72ºC for 10:00 8ºC forever END Overnight growth Inoculate 4 bottles of 10ml LB broth containing 10µl Kanamycin with colonies pUA66 Add 10µl Kanamycin 50mg/ml to each bottle of LB-broth Swab and add 1 colony of pUA66 from plate Place in shaker overnight at 37ºC Transformation of Ligation Perform at total of 6 transformation which includes self-ligation and viability test. Before: Place 2 bottles of 1ml SOC in a 37ºC incubator Collect 6 bottles of competent cell from the -80ºC freezer and place on ice for 5 minutes Add the plasmid / ligation product Tube A1, add the complete ligation reaction pUA66katE#A1 Tube A2, add the complete ligation reaction pUA66katE#A2 Tube B1, add the complete ligation reaction pUA66katE#B1 Tube B2, add the complete ligation reaction pUA66katE#B2 Tube S (Self-ligation test), add 2µl of digested plasmid pUA66#A1 Viability Test, add 1µl of pUA66 undigested plasmid Wait for 5 minutes to allow mixture to settle Place each tube in a 42ºC water bath for 1 minutes Place the cells back on ice and wait 5 minutes For each tube add 250µl SOC (warm 37ºC) Place the tubes in a shaker at 37ºC for 1 hour Pour the complete mixture onto plates containing Kanamycin Spread using glassbeads Incubate overnight at 37ºC