Tuesday
PCR
1)Prepare a 0.2 ml centrifuge tube for each sample.
2)Add reagent as Table 1.
Table 1 Reaction system (50μl total)
Components
Volumes (μl)
Template (human genomics DNA)
1
10×Taq buffer
5
MgCl2
4
10 mM dNTP
1
Forward primer (10uM)
1
Reverse primer (10uM)
1
Taq DNA polymerase
1
ddWater
36
3)Set program as Table 2 on PRC instrument.
Table 2 Reaction procedure
Step
Temperature, oC
time
Number of cycles
Initial denaturation
95
5min
1
Denaturation
95
30s
30
Annealing
54
30s
Extension
72
1min
Final elongation
72
10min
1
Final hold
4
forever
1
4)Verify the PCR product (~1000bp) by electrophoresis.
Plasmid Purification
1)Assemble a column stack by placing a Spin Column CP3 into a collection tube. Add 500 μl 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 and spin at 12000 rpm for1 min at room temperature. Discard supernatant.
3)Resuspend the bacterial pellet in 250 μl of Buffer P1. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
4)Add 250 μl of Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4–6times, and incubate at room temperature for 2 min.
Do not vortex, as this will result in shearing of genomic DNA.
5)Add 250 μl of Buffer P3, mix immediately and thoroughly by vigorously inverting 4–6 times. Centrifuge at 14000 rpm for 10 min.
6)Transfer supernatant to CP3 promptly. 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 μl Washing Buffer PW to CP3 and centrifuge at 12000 rpm for 1 min. Discard the liquid in collection tube. And replace 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 μl of Nuclease-Free Water to the CP3 and incubate at room temperature for 2 min.
11)Centrifuge at 12000 rpm for 2 min.
Nanodrop Analysis
For DNA
1)Raise the sampling arm and pipette the 2 μl 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. clone
3)Select the Sample Type to be measured from the Type drop-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 μl water again. Click the Blank.
5)Simply wipe the upper and lower pedestals. Pipette the 2 μl water onto the measurement pedestal. And click Measure.
6)Simply wipe the upper and lower pedestals and Pipette next sample, click measure again.
7)Clean with 2 μl water after use and make sure to place cover back on.
Agarose Gel Electrophoresis
Preparation of agorose gel:
Agarose
1×TAE
EB
DNA Fragment
0.8% Gel
0.2g
25 ml
2 μl
500bp~15kb
1% Gel
0.25 g
25 ml
2 μl
250bp~12kb
1.2% Gel
0.3 g
25ml
2 μl
150 bp~6kb
1.5%Gel
0.375g
25ml
2ul
80bp~4kb
1 g per 100 ml = 1% gel
1)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 see 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 swi
rling. Continue this cycle until there is no sign of any solid bits of agarose remaining.
5)Add 2 μl Ethidium bromide (EB) to 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 don’t 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 has solidified, the comb is removed.
Loading Samples and Running an Agarose Gel
1)Add loading buffer to each sample. eg. 1 μl of 6 × loading buffer to 1-2 μl plasmind or 5 μl PCR production.
Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and will also allows you to gauge how far the gel has run while you are running yo
ur 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.

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