Team:Exeter/lab book/gibs/wk3

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ExiGEM2012 Lab Book Gibs wk3


Single Gene Plasmids and Enzyme Characterisation \| Showcasing Polysaccharide Production \| The 3-Gene Inducible Plasmid Operon Construction \| Glycobase
9th - 13th July - 16th - 20th July - 23rd - 27th July - 30th July - 3rd August - 6th - 10th August - 13th - 17th August - 20th - 24th August - 27th - 31st August - 3rd - 7th September - 10th - 14th September - 17th - 21st September
	Operon Construction: 23rd - 27th July 2012 24.7.12 Mary, Freddie, Becca and Alex B. 9am 3A assembly of Pbad weak-RBS, Pbad strong-RBS and PlacI-RBS Upstream Pbad W (211.4 ng/µl) Pbad S (130.4 ng/µl) PlacI (299.2 ng/µl) MM DNA (500ng) 2.37 µl 3.83 µl 1.67 µl - EcoRI-HF 0.5 µl 0.5 µl 0.5 µl 2 µl SpeI 0.5 µl 0.5 µl 0.5 µl 2 µl 10X NEBuffer 2 2.5 µl 2.5 µl 2.5 µl 10 µl 100X BSA 0.25 µl 0.25 µl 0.25 µl 1 µl H2O (to 25 µl) 18.88 µl 17.42 µl 19.58 µl - Downstream RBS (77.4 ng/µl) DNA (500ng) 6.46 µl XbaI 0.5 µl PstI 0.5 µl 10X NEBuffer 2 2.5 µl 100X BSA 0.25 µl H2O to 25 µl 14.79 µl Destination plasmid MM DNA 250ng - EcoRI-HF 0.5 µl 2 PstI 0.5 µl 2 10X NEBuffer 2 2.5 µl 10 100X BSA 0.25 µl 1 H2O to 25 µl Incubated 36 °C for 10 minutes Heat inactivated at 80 °C for 20 minutes Ligation: Upstream part digest 2 µl Downstream part digest 2 µl Destination plasmid digest 2 µl 10X T4 DNA ligase buffer 2 µl T4 DNA ligase 1 µl H2O 11 µl Incubated at room temperature for 10 minutes Heat inactivated at 80 °C for 20 minutes Stored at -20°C prior to afternoon transformation 2.45pm Resuspended DNA according to IDT resuspension protocol (Alex B. and Becca then transformed them 2 µl of DNA from Pbad S-RBS and PlacI-RBS ligations added to 25 µl top10 competent cells Incubated on ice for 30 minutes Heat shocked 42 °C 30 seconds Placed on ice for 2 minutes Aseptically added 250 µl S.O.C. medium (pre-warmed) Shaken at 220 rpm, 37 °C, 1 hour Spread plates made 25.7.12 Mary and Freddie 9.45 am Biobricking attempt 2 To make Pbad Strong-RBS and PlacI-RBS 3A assembly Upstream Pbad S (130.4 ng/µl) PlacI (299.2 ng/µl) MM DNA (500ng) 3.8 µl 1.7 µl - EcoRI-HF 1 µl 1 µl 3 µl SpeI 1 µl 1 µl 3 µl 10X NEBuffer 2 5 µl 5 µl 15 µl 100X BSA 0.5 µl 0.5 µl 1.5 µl H2O (to 50 µl) 38.7 µl 40.8 µl - Downstream RBS (77.4 ng/µl) MM DNA (500ng) 6.5 µl - XbaI 0.5 µl 3 PstI 0.5 µl 3 10X NEBuffer 2 2.5 µl 15 100X BSA 0.25 µl 1.5 H2O 6.7 µl - Destination plasmid (tetracycline) pSB1T3 (linear) MM DNA 250ng 20 µl - EcoRI-HF 1 µl 3 PstI 1 µl 3 10X NEBuffer 2 5 µl 15 100X BSA 0.5 µl 1.5 H2O to 50 µl 22.5 µl - Incubated 36 °C for 10 minutes Heat inactivated at 80 °C for 20 minutes Ligation: Upstream part digest 2 µl Downstream part digest 2 µl Destination plasmid digest 2 µl 10X T4 DNA ligase buffer 2 µl T4 DNA ligase 1 µl H2O 11 µl Incubated at room temperature for 10 minutes Heat inactivated at 80 °C for 20 minutes Stored at -20°C prior to afternoon transformation Standard assembly run in parallel to 3A Upstream Pbad S PlacI MM DNA (500ng) 15.3 µl 6.7 µl - EcoRI-HF 1 µl 1 µl 3 µl SpeI 1 µl 1 µl 3 µl 10X NEBuffer 2 5 µl 5 µl 15 µl 100X BSA 0.5 µl 0.5 µl 1.5 µl H2O (to 40 µl) 17.2 µl 25.8 µl - Downstream RBS 2 (77.4 ng/µl) RBS 4 (63.0 ng/µl) MM DNA (500ng) 25.8 µl 31.7 µl - EcoRI-HF 1 µl 1 µl 3 XbaI 1 µl 1 µl 3 10X NEBuffer 2 5 µl 5 µl 15 100X BSA 0.5 µl 0. 5 µl 1.5 H2O 6.7 µl 0.8 - Digested for 1 hour at 36 °C DNA cleanup from enzymatic reactions using microcentrifuge and Quiagen column For RBS 2 and RBS 4 plasmids (post-digestion) 40 µl digest + 120 µl QG buffer + 40 µl isopropanol mixed and transferred to column (n.b. RBS 2 may not have been adequately mixed before being transferred to column) Centrifuged for 1 minute, 13000 rpm, room temp. Flow through discarded 500 µl buffer QG added to column Centrifuged for 1 minute, 13000 rpm, room temp. Flow through discarded 750 µl buffer PE added to column Centrifuged for 1 minute, 13000 rpm, room temp. Flow through discarded Centrifuged for 1 minute, 13000 rpm, room temp. Moved column to fresh 1.5 ml eppendorf 30 µl water added Left to stand for 1 minute Centrifuged 13000 rpm, 1 minute, room temp. DNA stored at -20 °C after analysis with a nanodrop spectrophotometer Transformation of 3A assembled Pbad strong-RBS and PlacI-RBS 2 µl DNA added to 50 µl top10 competent cells Incubated on ice 30 minutes Heat shocked 42 °C, 30 seconds Ice 2 minutes Aseptically added 250 µl S.O.C. medium Shaken 220 rpm, 37 °C, 1 hour Spread plates of 20 µl and 100 µl made on LB(tetracycline) plates Plates incubated at 37 °C overnight 26.7.12 Suspended sequencing primers (according to manufacturer’s instructed volumes) and prepared 50 µl aliquots for later Resuspended BBa_I0500 (large pbad promoter) in 10 µl water Left to stand for 5 minutes Transferred to eppendorf Centrifuged for 5 secs Transferred 2 µl to 50 µl top10 cells (remaining DNA stored at -20 °C) Incubated on ice 30 minutes Heat shocked 42 °C, 30 seconds Ice 2 minutes Aseptically added 250 µl S.O.C. medium Shaken 220 rpm, 37 °C, 1 hour Spread plates of 20 µl and 100 µl made on LB(kanamycin) plates Plates incubated at room temperature over weekend Transferred colonies from plates prepared 25.7.12 into 5 ml LB(tetracycline) broth (1:1000) Incubated overnight at 220 rpm, 37 °C 27.7.12 Miniprep of 3A assembled plasmid (from 25.7.12) Transferred broth to fresh falcon tubes to remove pipette tips (tips and original tubes stored in fridge until same day streak-plates made) Centrifuged 5 minutes, 3901 rcf, 4 °C Supernatant discarded 250 µl resuspension solution added Pipetted to mix and transferred to clean 1.5 ml eppendorf 250 µl lysis solution added Inverted ~6 X 350 µl neutralisation solution added Inverted ~6 X Centrifuged 5 mins, 13000 rpm, room temperature Transferred supernatant to spin column Centrifuged for 1 minute, 13000 rpm, room temperature Discarded flow-through 500 µl wash solution added Centrifuged 1 minute, 13000 rpm, room temperature Discarded flow-through 500 µl wash solution added Centrifuged 1 minute, 13000 rpm, room temperature Discarded flow-through Centrifuged 1 minute, 13000 rpm, room temperature Transferred column to clean 1.5 ml eppendorf 50 µl water added Centrifuged 1 minute, 13000 rpm, room temperature 5 µl water added Centrifuged 1 minute, 13000 rpm, room temperature Column discarded, DNA concentration measured and DNA stored in eppendorf at -20 °C PlacI-RBS 1. 110.5 ng/µl 2. 214.9 ng/µl 3. 182.2 ng/µl 4. 259.1 ng/µl Digest for gel electrophoresis analysis 1 2 3 4 master mix Plasmid DNA 18.1 9.3 11 7.7 - EcoRI-HF 1 1 1 1 5 PstI 1 1 1 1 5 10X NEBuffer 2 5 5 5 5 25 100X BSA 0.5 0.5 0.5 0.5 2.5 H2O 0 8.2 6.5 9.8 - Digested at 36 °C for 30 minutes Streak plates made from dregs of overnight culture (see above) >