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 Tuesday 24.7.12 Mary, Freddie, Becca and Alex B. 9am 3A assembly of Pbad weak-RBS, Pbad strong-RBS and PlacI-RBS Upstream For 500ng DNA: Pbad Weak - (211.4 ng/µl)=2.37 µl Pbad Strong -(130.4 ng/µl) =3.83 µl PlacI - (299.2 ng/µl)=1.67 µl EcoRI-HF: 0.5 µl (master mix volume 2 µl) SpeI: 0.5 µl (master mix volume 2 µl) 10X NEBuffer 2: 2.5 µl (master mix volume 10 µl) 100X BSA: 0.25 µl (master mix volume 1 µl) H2O (to 25 µl): Pbad Weak =18.88 µl Pbad Strong =17.42 µl PlacI =19.58 µl Downstream For 500 ng DNA: RBS (77.4 ng/µl)=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 Linear plasmid DNA (250ng): 20 µl EcoRI-HF: 0.5 µl (master mix vol. 2 µl) PstI: 0.5 µl (master mix vol. 2 µl) 10X NEBuffer 2: 2.5 µl (master mix vol. 10 µl) 100X BSA: 0.25 µl (master mix vol. 1 µl) 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) 1.2 µl of DNA from Pbad S-RBS and PlacI-RBS ligations added to 25 µl top10 competent cells 2.Incubated on ice for 30 minutes 3.Heat shocked 42 °C 30 seconds 4.Placed on ice for 2 minutes 5.Aseptically added 250 µl S.O.C. medium (pre-warmed) 6.Shaken at 220 rpm, 37 °C, 1 hour 7.Spread plates of 20 and 100 µl made 8.incubated at 37 °C overnight Wednesday 25.7.12 Mary and Freddie 9.45 am Biobricking attempt 2 To make Pbad Strong-RBS and PlacI-RBS (n.b. placI-RBS relevant only to 3 gene part of project) 3A assembly Upstream For 500ng DNA: Pbad Strong -(130.4 ng/µl) =3.8 µl PlacI - (299.2 ng/µl)=1.7 µl EcoRI-HF: 1 µl (master mix vol. 3 µl) SpeI: 1 µl (master mix vol. 3 µl) 10X NEBuffer 2: 5 µl (master mix vol. 15 µl) 100X BSA: 0.5 µl (master mix vol 1.5 µl) H2O (to 50 µl): Pbad strong: 38.7 µl PlacI: 40.8 µl Downstream For 500 ng DNA: RBS (77.4 ng/µl)=6.5 µl XbaI: 0.5 µl (master mix vol. 3 µl) PstI: 0.5 µl (master mix vol. 3µl) 10X NEBuffer 2: 2.5 µl (master mix vol. 15 µl) 100X BSA: 0.25 µl (master mix vol. 1.5 µl) H2O: 6.7 µl Destination plasmid (pSB1T3 - tetracycline resistance) Linear plasmid DNA (250ng): 20 µl EcoRI-HF: 1 µl (master mix vol. 3 µl) PstI: 1 µl (master mix vol. 3 µl) 10X NEBuffer 2: 5 µl (master mix vol. 15 µl) 100X BSA: 0.5 µl (master mix vol. 1.5 µl) 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: DNA (500ng): Pbad Strong: 15.3 µl PlacI :6.7 µl EcoRI-HF: 1 µl (master mix vol. 3 µl) SpeI: 1 µl (master mix vol. 3 µl) 10X NEBuffer 2: 5 µl (master mix vol. 15 µl) 100X BSA: 0.5 µl (master mix vol. 1.5 µl) H2O (to 40 µl): Pbad Strong: 17.2 µl PlacI : 25.8 µl Downstream: DNA (500ng) RBS 2 (77.4 ng/µl): 25.8 µl RBS 4 (63.0 ng/µl): 31.7 µl - EcoRI-HF: 1 µl (master mix vol. 3 µl) XbaI: 1 µl (master mix vol. 3 µl) 10X NEBuffer 2: 5 µl (master mix vol. 15 µl) 100X BSA: 0.5 µl (master mix vol. 1.5 µl) H2O : RBS 2: 6.7 µl RBS 4: 0.8 Digested for 1 hour at 36 °C DNA cleanup from enzymatic reactions using microcentrifuge and Quiagen column (n.b. this was intended for gel electrophoresis of upstream fragments to be run in parallel, but the gel was unsuccessful. It later turned out we had the wrong sequences and had not realised the sequences were too short to show on a gel for gel purification.) For RBS 2 and RBS 4 plasmids (post-digestion) 1.40 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) 2.Centrifuged for 1 minute, 13000 rpm, room temp. 3.Flow through discarded 4.500 µl buffer QG added to column 5.Centrifuged for 1 minute, 13000 rpm, room temp. 6.Flow through discarded 7.750 µl buffer PE added to column 8.Centrifuged for 1 minute, 13000 rpm, room temp. 9.Flow through discarded *10.Centrifuged for 1 minute, 13000 rpm, room temp. 11.Moved column to fresh 1.5 ml eppendorf 12.30 µl water added 13.Left to stand for 1 minute 14.Centrifuged 13000 rpm, 1 minute, room temp. 15.DNA stored at -20 °C after analysis with a nanodrop spectrophotometer Transformation of 3A assembled Pbad strong-RBS and PlacI-RBS 1.2 µl DNA added to 50 µl top10 competent cells 2.Incubated on ice 30 minutes 3.Heat shocked 42 °C, 30 seconds 4.Ice 2 minutes 5.Aseptically added 250 µl S.O.C. medium 6.Shaken 220 rpm, 37 °C, 1 hour 7.Spread plates of 20 µl and 100 µl made on LB(tetracycline) plates 8.Plates incubated at 37 °C overnight Thursday 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 - selected as alternative to pbad strong due to sequence match with Gibson primers) in 10 µl water Left to stand for 5 minutes Transferred to eppendorf Centrifuged for 5 secs 1.2 µl DNA added to 50 µl top10 competent cells (remaining DNA stored at -20 °C) 2.Incubated on ice 30 minutes 3.Heat shocked 42 °C, 30 seconds 4.Ice 2 minutes 5.Aseptically added 250 µl S.O.C. medium 6.Shaken 220 rpm, 37 °C, 1 hour 7.Spread plates of 20 µl and 100 µl made on LB(kanamycin) plates 8.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 Friday 27.7.12 9 am Miniprep of 3A assembled plasmid (from 25.7.12) 1.Transferred broth to fresh falcon tubes to remove pipette tips (tips and original tubes stored in fridge until same day streak-plates made) 2.Centrifuged 5 minutes, 3901 rcf, 4 °C 3.Supernatant discarded 4.250 µl resuspension solution added 5.Pipetted to mix and transferred to clean 1.5 ml eppendorf 6.250 µl lysis solution added 7.Inverted ~6 X 8.350 µl neutralisation solution added 9.Inverted ~6 X *10.Centrifuged 5 mins, 13000 rpm, room temperature 11.Transferred supernatant to spin column 12.Centrifuged for 1 minute, 13000 rpm, room temperature 13.Discarded flow-through 14.500 µl wash solution added 15.Centrifuged 1 minute, 13000 rpm, room temperature 16.Discarded flow-through 17.500 µl wash solution added 18.Centrifuged 1 minute, 13000 rpm, room temperature 19.Discarded flow-through 20.Centrifuged 1 minute, 13000 rpm, room temperature 21.Transferred column to clean 1.5 ml eppendorf 22.50 µl water added 23.Centrifuged 1 minute, 13000 rpm, room temperature 24.5 µl water added 25.Centrifuged 1 minute, 13000 rpm, room temperature 26.*Column discarded, DNA concentration measured and DNA stored in eppendorf at -20 °C (n.b. digest gel was run for analysis, but for this part of the project is considered irrelevant due to inconclusive results from small fragment size and because BBa-I0500 was decided to be more useful for this part of the project) Streak plates made from dregs of overnight culture (see above)