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Lab Protocols Overview of Molecular Cloning

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Lab Protocols Overview of Molecular Cloning Lab Protocols Overview of Molecular Cloning pZ Plasmid System ​ Nucleic Acids Res. 1997 Mar 15;25(6):1203­10. ​ Expanded Naming Set of pZ plasmids (Tal Danino, 4/27/2011) Sm = pSC101m (Jesse's higher copy variation , 10­15 copies)(note this looks more like pSC101 than pSC101*) 5a = Lux/Tet Hybrid Promoter 7=Parsenic promoter 8=PL promoter (lambda) QIAprep Miniprep [Designed for the purification of up to 20 µg high­copy plasmid DNA from 1­5 ml overnight E.coli culture in LB medium] ​ ​ 14mL culture tube protocol: (Convenient when doing many minipreps) 1. Spin down culture tubes in big centrifuge for 5 mins. [14mL culture tubes can be spun down in big centrifuge up to 3g] 2. Pour supernatant (&tip) into bleach flask, being careful to remove all liquid. If liquid remains in tube then use pipette to remove excess. 3. Add 250 uL buffer P1 to each tube. 4. Place tubes on rack and vortex entire rack slowly till resuspended (watch that fluid doesn't reach top of tube, usually setting 4­6) 5. Transfer 300uL to 1.5 mL microcentrifuge tube. This replaces steps 1­4 below. Original Protocol: ∙ NOTE: Use Blue spin columns; centrifuge rpm 14000 ​ ​ 1. Pipette 1.5ml from the test tube used for growth and transfer to clean 1.5ml microcentrifuge tube. 2. Spin down cells for 9­10 seconds @ 14000 rpm and remove LB medium. 3. Add 1.5 mL of culture again & repeat. 4. Re­suspend pelleted bacterial cells (via vortexing) in 200 µl Buffer P1 (located in right­most fridge) 5. Add 250 µl Buffer P2 and mix gently by inverting tube 4­6 times. Solution should turn blue. 6. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4­6 times. Solution should turn colorless. 7. Centrifuge for 10 min. 8. Pipette (700ul) the supernatant into the QIAprep spin column. 9. Centrifuge for 60 sec. Discard the flow­through. 10. Wash QIAprep by adding 0.70 ml (700 µl) Buffer PE and centrifuge 60 sec. 11. Discard the flow­through, and centrifuge for an additional 2 min to remove residual wash buffer. Wait 2­3 mins to dry (removes excess PE buffer and gives better sequencing results). 12. To elute DNA, place QIAprep column in a clean microcentrifuge tube. Add 30 µl Buffer EB to center of each spin column, let stand for 1 min, and centrifuge for 1 min. 13. NanoDrop to find concentration. High volume minipreps: If plasmid is low copy (i.e. SC101) or a high amount/concentration is ​ desired, grow 50 mL of cell cultures and use this modified miniprep protocol: I typically only do 10mL of culture per tube to allow for clean minipreps. In the past I've had trouble sequencing preps with too much culture volume, or I see very high bands of DNA on my gels (probably genomic DNA). ­­ For 50mL's culture A) Spin down in big centrifuge for 5 mins, remove media thoroughly B) Add 7.5x amount of P1 (1875 uL) C) Resuspend, and aliquot to 5 tubes (400 uL each) D) Add 1.5x amount of P2 (375uL), invert gently 4­6 times E) Add 1.5x amount of P3 (525uL), invert throughly F) Spin Down for 10 mins G) Run supernatant through 3­5 columns H) Elute with 50­100uL Notes: Also, after step (F), I sometimes collect the supernatant of microcentrifuge tubes into a big tube to prevent contamination with cell debris. Sometimes I do PE buffer wash twice when a high culture volume is used as well. NanoDrop Spectrophotometer Designed to find the concentration (in ng/µl) of DNA. Procedure: ∙ NOTE: Use 2 µl of liquid for each measurement ​ ​ 1. Blank the instrument using Buffer EB. 2. Drop liquid onto meter and press “measure.” 3. Write concentration on side of microcentrifuge tube – “[concentration]”. 4. Wipe liquid off of bottom­ and top­half of meter using Kimwipe. 5. Repeat steps 2­4 for each solution. Agarose Gel Electrophoresis Designed to separate DNA, RNA, or protein molecules using an electric current applied to a gel matrix. “Creating the Gel” Procedure: ∙ CAUTION: Ethidium Bromide (EtBr) is a carcinogen. When handling, be sure to ​ ​ wear gloves and clean any spills thoroughly. 1. Weigh 0.35 g of agarose powder and pour into “EtBr” flask (For a standard 0.7% w/v gel) 2. Measure 50 ml of Buffer TAE and pour into “EtBr” flask. 3. Heat “EtBr” flask in lab microwave for 1 min 15 sec, or until all agarose particles dissolve. 4. Remove flask from microwave using “hot hands” and swirl (careful for vigorous bubbling). 5. Take plastic gel tray and ensure it is tight on gel caster. Insert desired gel comb into grooves. 6. Pipette 5 µl of GelRed dye into flask and swirl to ensure uniformity. 7. Pour contents of flask into plastic gel tray and let stand for ~30 min to solidify. “Running the Gel” Procedure: ∙ NOTE: Loading dye is “6x,” meaning if there is 5 µl of DNA to be tested, 1 µl of dye ​ ​ should be added. Ladder used is 1kb (1000 base pairs). Both dye and ladder are located in third­to­right fridge. ∙ NOTE: Gel may be run @ 110 volts for 40 min(gel extractions, more careful gels) ​ ​ or @ 150 volts for 20 min (diagnostics/quick checks) 1. Remove tape and well placers from gel tray. 2. Place gel tray into electrophoresis apparatus, ensuring that Buffer TAE covers the gel entirely. 3. Mix liquid DNA solution and dye. Solution should turn blue/violet. 4. Pipette liquid (generally 10­15 µl) into respective wells. Begin and end with ladder. 5. Place cover onto electrophoresis apparatus, matching red to red, black to black, and press “run.” 1kb Ladder ­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­ 2 Log ladder “Assessing the Results” Procedure: ∙ CAUTION: Be sure to turn off UV lamp when done with photo­capture machine. If ​ ​ gel is to be saved for extraction, limit gel exposure to UV light. ∙ NOTE: Gel will be prone to slide off tray when wet. ​ ​ 1. Remove gel tray from electrophoresis apparatus, letting buffer drip off. 2. Wipe bottom of gel tray and place in photo­capture machine (located near lab microwave). 3. Focus and adjust light (white) of photo. 4. Turn on UV lamp and capture photo. 5. Press save and dump gel into designated bucket located to right of machine. 6. Access and print the photo through server online at ftp://toggle.ucsd.edu. Label wells and compare results with the ladder in order to designate size of DNA fragments. Special Considerations: For high separation of small fragments in the range of 0­1000bp, use a 1% gel. For separation of large fragments use a 0.5% gel. Gel Digests To cut DNA at specific sequences and often to leave sticky ends for ligating pieces together. There are two types of Digests we do with restriction enzymes: (1) screening/diagnostic of colonies (2) digesting PCRs/plasmids for gel extractions&ligations. (1) For diagnostic gels, we use between 50­200ng of DNA and digest for 1/2­1 hour before running on a gel. This is just to check if our DNA has the correct fragments (2) For gel extractions, we use between 1500­3000ng of DNA and digest for 2­3 hours before running on a gel. The gel extraction procedure has low yield thus a lot is needed to start with. Type (1) Diagnostic Gels ∙ MIX: Typically 1.0­4.0 microliters DNA, 1.0 of each 10x buffer (check chart), ​ 0.25­0.5 of each enzyme, fill up to 10.0 microliters total with water. ∙ Leave at 37 incubator for 1/2­1 hour ​ ∙ Make gel with thin comb ​ ∙ By the time gel solidifies, probably ready to run gel ​ Typical Mix is 2.0 DNA 1.0 Buf 2 1.0 BSA 0.5 KpnI 5.5 H20 10.0 TOTAL Type (2) Gel Extractions ∙ Digest desired amount of DNA (will probably be around 30­60 microliters) for 2­3 ​ hours at 37. ∙ Make wide gel comb ​ ∙ Run gel till bands are well separated and cut out gel piece, trying to minimize ​ amount of agarose Typical Mix 46.0 DNA 6.0 Buf 2 6.0 BSA 1.0 KpnI 1.0 MluI 60.0 TOTAL Note: After creating mix, vortex briefly, then spin down briefly to ensure consistency. Note: Addition of BSA does not affect digests so if 1 enzyme requires it, just add it. ­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­ Creating Master Mixes Often times a large number of samples are digested with the same enzymes & buffers, etc., so a mix is created for all of them, then aliquoted into the tubes. This is to ensure uniformity across samples and allow for ease of pipetting. For instance if your gel digest mix is: 2.5 DNA 1.0 Buf 2 0.25 KpnI 6.25 H20 and you need this for 9 different samples of DNA, then your master mix is created for 10 samples without the DNA in it (always +1 or +2 the # of samples) : 10.0 Buf 2 2.5 KpnI 62.5 H20 =75 / 10=7.5 microliters/reaction Before using, vortex & mini­centrifuge your mix to get the liquid to the bottom & ensure it is well mixed. Keep on ice until aliquoted. The same technique is done for PCR mixes. ­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­ Gel Purification ​ Designed to purify nucleic acids from an excised gel fragment 1. Weigh excised DNA (TARE with blank tube) 2. Add 3 gel volumes buffer QG(i.e. if piece is 100mg, add 300uL QG). 3. Allow to dissolve at 50 degrees for ~10 mins, vortexing every few mins 4.
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