分子克隆操作流程英文版Clone Experimental Protocol

1、.TuesdayPCR1) Prepare a 0.2 ml centrifuge tube for each sample.2) Add reagent as Table 1.Table 1 Reaction system (50l total)ComponentsVolumes (l)Template (human genomics DNA)110×Taq buffer5MgCl2410 mM dNTP1Forward primer (10uM)1Reverse primer (10uM)1Taq DNA polymerase1ddWater363) Set program as

2、 Table 2 on PRC instrument.Table 2 Reaction procedureStep Temperature, oCtimeNumber of cyclesInitial denaturation955min1Denaturation9530s30Annealing5430sExtension721minFinal elongation7210min1Final hold4forever14) Verify the PCR product (1000bp) by electrophoresis.Plasmid Purification1) Assemble a c

3、olumn stack by placing a Spin Column CP3 into a collection tube. Add 500 l Balance Liquid (BL) to Spin Column CP3 and spin at 12000 rpm for1 min at room temperature. Discard the liquid in collection tube. And replace CP3 to collection tube.2) Transfer 1 ml bacterial cells to 1.5 ml centrifuge tube a

4、nd spin at 12000 rpm for1 min at room temperature. Discard supernatant.3) Resuspend the bacterial pellet in 250 l of Buffer P1. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.4) Add 250 l of Buffer P2, mix thoroughly by vigorously inve

5、rting the sealed tube 46times, and incubate at room temperature for 2 min. Do not vortex, as this will result in shearing of genomic DNA.5) Add 250 l of Buffer P3, mix immediately and thoroughly by vigorously inverting 46 times. Centrifuge at 14000 rpm for 10 min. 6) Transfer supernatant to CP3 prom

6、ptly. Do not allow the pelleted debris to fall into the column. . Centrifuge at 12000 rpm for 1 min. Discard the liquid in collection tube. And replace the CP3 to collection tube.7) Add 600 l Washing Buffer PW to CP3 and centrifuge at 12000 rpm for 1 min. Discard the liquid in collection tube. And r

7、eplace the CP3 to collection tube.8) Repeat steps 7.9) Centrifuge at 12000 rpm for 2 min, and air-dry for 10 min.10) Insert the CP3 into a 1.5 ml centrifuge tube. Add 35 l of Nuclease-Free Water to the CP3 and incubate at room temperature for 2 min.11) Centrifuge at 12000 rpm for 2 min.Nanodrop Anal

8、ysisFor DNA1) Raise the sampling arm and pipette the 2 l water onto the lower measurement pedestal.2) Select the Nucleic Acid application from the main menu. If the wavelength verification window appears, ensure the arm is down and click OK. 3) Select the Sample Type to be measured from the Type dro

9、p-down list. The default setting is DNA-50. Simply wipe the upper and lower pedestals using a dry laboratory wipe and the instrument is ready to measure the next sample.4) Pipette the 2 l water again. Click the Blank.5) Simply wipe the upper and lower pedestals. Pipette the 2 l water onto the measur

10、ement pedestal. And click Measure.6) Simply wipe the upper and lower pedestals and Pipette next sample, click measure again.7) Clean with 2 l water after use and make sure to place cover back on.Agarose Gel ElectrophoresisPreparation of agorose gel:Agarose1×TAEEBDNA Fragment0.8% Gel0.2g25 ml2 l

11、500bp15kb 1% Gel0.25 g25 ml2 l250bp12kb 1.2% Gel0.3 g25ml2 l150 bp6kb1.5%Gel0.375g25ml2ul80bp4kb1 g per 100 ml = 1% gel1) Weigh out the required quantity of agarose powder. Place it into an Erlenmeyer flask.2) Add the appropriate quantity of 1×TAE.3) Microwave on high just until you start to se

12、e the appearance of boiling. Remove the flask. Carefully, swirl the agarose mixture. 4) Return the flask the microwave. Microwave it again until you see boiling. Repeat the swirling. Continue this cycle until there is no sign of any solid bits of agarose remaining.5) Add 2 l Ethidium bromide (EB) to

13、 the liquid gel. Ethidium bromide, a fluorescent dye used for staining nucleic acids.6) Allow the agarose to cool to 50 oC to 65 oC. If you dont it will warp the gel boxes. 7) Pour the agarose into a gel tray with the well comb in place and allowed to solidify at room temperature.8) After the gel ha

14、s solidified, the comb is removed.Loading Samples and Running an Agarose Gel1) Add loading buffer to each sample. eg. 1 l of 6 × loading buffer to 1-2 l plasmind or 5 l PCR production.Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and will als

15、o allows you to gauge how far the gel has run while you are running your gel; and 2) it contains a high % glycerol, so after adding it your sample is heavier than water and will settle to the bottom of the gel well, instead of diffusing in the buffer.2) Once solidified, place the agarose gel into th

16、e gel box (electrophoresis unit).3) Fill gel box with 1×TAE until the gel is covered.4) Carefully load 3 l DNA ladder into the first lane of the gel. eg. 100 bp DNA ladder for PCR production. 1000 bp DNA ladder for plasmid.5) Carefully load your samples into the additional wells of the gel.6) R

17、un the gel at 80-150V until the dye line is approximately 75-80% of the way down the gel. Note: Black is negative, red is positive. (The DNA is negatively charged and will run towards the positive electrode.) Always Run to Red.7) Using any device that has UV light, visualize your DNA fragments.Analy

18、zing Your Gel:Using the DNA ladder in the first lane as a guide (the manufacturer's instruction will tell you the size of each band), you can interpret the bands that you get in your sample lanes to determine if the resulting DNA bands that you see are as expected or not.Purification of PCR Prod

19、ucts1) Column Balancing: put the CB2 column into a collection tube, and add 500ul BL, centrifuge 1 min at 12000rpm, discard the liquid in the collection tube, and then put the CB2 back to collection tube.2) Add 5 volumes buffer PB into PCR products.3) Add the solution in step 2 into a balanced colum

20、n, incubate in room temperature for 2 min, then centrifuge 1 min at 12000 rpm, discard the liquid in the collection tube.4) Add 600ul buffer PW, centrifuge 1 min at 12000 rpm, discard the liquid in the collection tube, and then put the CB2 back to collection tube.5) Repeat step 4.6) Put the CB2 back

21、 to collection tube, centrifuge 2 min at 12000 rpm to remove PW completely. Open the lid of CB2 for several minutes to dry it. (It is recommened to warm a tube of ddwater while waiting column drying.)7) Put the CB2 column into a new 1.5ml centrifuge tube, add at least 30ul warmed water, incubate for

22、 2 min, and centrifuge 2 min at 12000rpm to collect the DNA.Double Cutting of PCR ProductsPrepare a 1.5 ml centrifuge tube for each sample.Reaction system (40ul total)ComponentsVolumes (ul)PCR product3010x Buffer2.14 XhoI1NheI1ddwater4Total40ComponentsVolumes (ul)pcDNA-GFP2ug10x Buffer2.14 XhoI1NheI

23、1ddwaterto 40ulTotal40ulAfter all components added, centrifuge the tubes and incubate them at 37 oC for 30 min.During the half hour, youd better prepare an agarose gel for gel extraction (tiangen kit).Transformation for Plasmid1) Take out the competent cells (DH5) from the -80 oC fridge, and put it

24、on the ice to thaw.2) Add approximately 50ng plasmid to 50ul competent cells, stay on ice for 30min.3) Heat-shock the cells for 60 seconds at 42°C without shaking. Then put them on ice 3min quickly.4) Use the glass bar to spread the competent cells evenly. 37 oC，Invert the selective plate(s) an

25、d incubate at 37 oC overnight5) Store the plate at 4°C.ThursdayDNA Gel Exaction1) Column Balancing: put the CA2 column into a collection tube, and add 500ul BL, centrifuge 1 min at 12000rpm, discard the liquid, and then put the CB2 back to collection tube.2) Cut the DNA band from the agarose gel, and then put it into the new tube (remember weigh it in advance), weigh them.3) Add the equal volume solution PN as the weigh of DNA band (if the DNA band is 0.1g, we can add 100ul solution PN). Then incubate them at 50 oC, confirm the gel is dissolve. 4) Add the solution(the room