ÐÏࡱá > þÿ Š Œ þÿÿÿ ˆ ‰ ÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿì¥Á %` ð¿ [L bjbj"x"x .P @ @ B ÿÿ ÿÿ ÿÿ ¤ $ ,- ,- ,- ,- L x- \ $ ’ 2 à- à- à- à- à- / / / á~ ã~ ã~ ã~ ã~ ã~ ã~ $ Ā h ,ƒ p  E =0 ë. " / =0 =0  à- à- L 6 6 6 =0 ^ à- à- á~ 6 =0 á~ 6 6 † ‘u Ð uz à- Ô- @Æ>L4É ,- ›3 ‚ aw 4 | Ì b 0 ’ •w à œƒ 4 𠜃 h uz œƒ uz   / > K/ , 6 w/ $ ›/ ¢ / / /   6 / / / ’ =0 =0 =0 =0 $ $ $ D h! Ä $ $ $ h! $ $ $ ÿÿÿÿ High Throughput Cloning-MCSG19 Manual KOD PCR set up Primer Plate Preparation: Combine and dilute stock primers-forward and reverse (50 µM). Add 1µl of forward and reverse stock primers to 38 µl of H2O to make 1.25 µM final concentration. Mix, quick spin and store at -20oC. Increased dilution plate: 5µl FP and 5µl RP plus 190µl H2O Always do a quick spin for the FR primer plates and then make the dilute mix. Template Plate Preparation: Make 1:25 dilution of template plasmid with TE. Store at -20oC. Quick spin template plasmid and quick spin again after dilution. Reaction set up: Vortex before adding (except KOD polymerase) Prepare reaction mix as follows from the KOD kit: 1 x 110 x H2O 26 µl 2860 µl 10x KOD Buffer 5 µl 550 µl dNTPs (2 mM each) 5 µl 550 µl MgSO4 (25 mM) 2 µl 220 µl KOD polymerase (1 U/µl) 1 µl 110 µl F/R primers (12.5 µM each) 10 µl Template 1µl Total vol. 50 µl 4290 µl Mix well. Transfer 39µl of the master mix to each well. Add 10 µl of the diluted F/R primers. Add 1 µl of diluted plasmid DNA. Centrifuge briefly to ensure liquid contents are at the bottoms of the wells. Start amplification. Cycling parameter: (2 hours) Denature 95oC 3 min Cycle 95oC 40 sec 53oC 40 sec 32 cycles 72oC 1.5 min Polish 72oC 10 min Hold 4oC Agarose gel Mix 5 µl of PCR reaction with 2 µl of 5x agarose gel loading dye. Mix and quick spin. Run it on a 1% agarose/TAE gel. (2g of agarose in 200 ml of 1x TAE with 7 µl of ethidium bromide). Heat for about 1.5-2 mins but when starts to boil mix it and heat it again. After it cools down, add ethidium bromide. 10X TAE buffer PCR clean up Clean up PCR reactions using Qiagen’s QIAQuick PCR Purification kit per manufacture’s protocol. Elution volume: 150 µl of TE. Cleaned PCR products can be store at -20oC. PicoGreen Take Picogreen dye out of the fridge and allow it to come to RT, protect it from light. Mix 55µl of Picogreen with 11 ml of TE. Use this mixture immediately as it is light sensitive. Transfer 3 µl of cleaned PCR product to 97 µl of H2O in a 96-well plate (optical plate for reading!). Add 100 µl of diluted Picogreen. Mix and read absorbance at 450 nm immediately. Password: mcsg#2007 LIC reaction set up Make LIC reaction master mix as follows for one plate: Vortex before adding. 10x LIC reaction buffer 465 µl dCTP (25 mM) 465 µl DTT (100 mM) 228 µl H2O 61 µl T4 DNA Polymerase (tap it) 250 U (100 µl) Keep the mix on ice. dCTP (25mM): 100mM available so 1:4 dilution (200µl dCTP + 600µl H2O) Transfer 16 µl of cleaned PCR products to a 96-well plate. Add 16 µl of H2O. Add 10.4 µl of above reaction mix. Pipet up and down several times to mix. (quick spin as well) Let reactions stand at RT for 30 min. Transfer LIC plate to PCR machine. Heat 20 min at 75oC to stop the reaction. The plates can be stored at -20oC. Annealing Reaction and Transformation Prepare a plate with 4 µl of LIC-treated vector (actual vol depends on the prep). pMCSG19 prepared by Wen use 16.5ul. Mix: 423.5 µl water + 16.5 µl vector (4µl to each well) Add 4 µl of LIC reaction of the PCR product to the plate. Mix (with pipet and quick spin) and stand at RT for 20 min. Add 45 µl of PRK1037 competent cells into the plate (competent cells were thawed on ice). Briefly centrifuge the plate to make sure the competent cells mix with the annealing reaction. And incubate the plate on ice for 5-15 min. After/during adding PRK1037 keep plate on ice! Heat shock the cells by heating the plate 1 min at 48oC on PCR machine (or a heat block). The plate is then incubated on ice for 2-10 min. Add 120 µl of LB (no amp or kan!) to each well. Incubate the plate at 37oC for 30 min. (no mixing) Plate out 50% glycerol: 50mL H2O+ 50mL 100% Glycerol. Autoclave it for 30 minutes. LB (12.5g in 500mL + 7.5g Agar) and autoclave it. When cool enough to touch add amp (500 µl of Amp, 500µl of Kan) Pour 500 ml LB agar with Amp into two grid plates. Plate out 150 µl of above recovered culture into each grid to ensure ~ 20 single colonies for each transformation. (plate out vol depends on LIC vector and competent cells efficiencies). Incubate the plates at 37oC overnight. Expression/Solubility assay Fill a deep well plate with 0.6 ml of LB/AMP/KAN (Can use 2XYT media) Pick colonies into deep well block and incubate at 37oC with shaking to an OD600 of 0.5. Take out 100 µl of culture in a PCR plate, add 100 µl of sterile 50% glycerol and store at -80oC. This is the frozen stock. Add IPTG to remaining 0.5 ml of culture, final concentration 1 mM. Continue incubating at 37oC for another 2 hours (or 20oC overnight depends on the schedule). 1000X-250X: Dilution of IPTG:110 µl of 1000X + 330 µl 2XYT (add 2 µl of IPTG to each well) Take 200 µl of culture in PCR plate (2X 100 µl). Spin down at 4000 rpm for 10 min at 4oC to pellet cells. Discard supernatant and resuspend cell pellets in 30 µl of SDS loading dye. Denature 5 min at 95-100oC and store at -20oC. This is to check expression of proteins. About 10 µl of each will be loaded on SDS-PAGE gel. Pellet the remaining 300 µl (2x 150ul into a PCR plate) of cultures by spinning as above. Cell pellets can be stored at -20oC or proceed to next step. Add 32 µl of lysis buffer to each sample(mix with pipettes)and incubate 20 minutes at RT. Freeze-thaw 3 times. Centrifuge 10 min at 4000 rpm at 4oC. Total Volume of Lysis BufferReagents2mL3mL4mL5mL6mL7mL8mL9mL10mL11mLH2O~2mL~3mL~4mL~5mL~6mL~7mL~8mL~9mL~10mL~11mL1M Pi buffer, pH 8 1 0 0 ¼l 1 5 0 ¼l 2 0 0 ¼l 2 5 0 ¼l 3 0 0 ¼l 3 5 0 ¼l 4 0 0 ¼l 4 5 0 ¼l 5 0 0 ¼l 5 5 0 ¼l 5 M N a C l 1 2 0 ¼l 1 8 0 ¼l 2 4 0 ¼l 3 0 0 ¼l 3 6 0 ¼l 4 2 0 ¼l 4 8 0 ¼l 5 4 0 ¼l 6 0 0 ¼l 6 6 0 ¼l P I 1 2 ¼l 1 8 ¼l 2 4 ¼l 3 0 ¼l 3 6 ¼l 4 2 ¼l 4 8 ¼l 5 4 ¼l 6 0 ¼l 6 6 ¼l L z 8 ¼l 1 2 ¼l 1 6 ¼l 2 0 ¼l 2 4 ¼l 2 8 ¼l 3 2 ¼l 3 6 ¼l 4 0 ¼l 4 4 ¼l B z 2 ¼l 3 ¼l 4 ¼l 5 ¼l 6 ¼l 7 ¼l 8 ¼l 9 ¼l 1 0 ¼l 1 1 ¼l P I : P r o t e a s e I n h i b i t o r C o c k t a i l ( S i g m a ) L z : r L y z o z y m e ( N o v a g e n ) B z : B e z o n a s e ( N o v a g e n ) T r a n s f e r 3 0 µ l o f s u p e r n a t a n t t o a n o t h e r p l a t e . A d d 1 0 µ l o f S D S l o a d i n g d y e t o t h e p l a t e containing the supernatant. Denature as above and store at -20oC. This is to check solubility of expressed proteins. 15 µl will be loaded on SDS-PAGE gel. 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