Difference between revisions of "GDSYZX-United/NOTEBOOK/procedure record"

Making the agarose gel

1.Experiment reagent : agarose, TAE solution, SYBY GREEN
2.Experiment apparatus:microwave oven, analytical balance, graduated cylinder100ml, conical flask500ml, plastic glove，the equipment for making gel(including the combs), electrophoresis tank
3.Experiment steps:
    (1)add agarose 0.8g, TAE solution 100μl into the conical flask
    (2)put a plastic glove on the conical flask (prevent it from evaporating)
    (3)put the conical flask into the microwave oven to heat up for 2min
    (4）take the conical flask out, after it is cooled down, add nucleic acid dye SYBY GREEN 120μl into it , and then shake it until it is mixed uniform
    (5)pour the solution into Agarose gel plate, until it is 4~6mm thick
    (6)put an suitable comb in the plate, and then cool the gel down in indoor temperature
    (7)after it is solidified completely, pull out the comb vertically to make sure the sample hole is all right
    (8)put the gel into the electrophoresis tank, add TBE until it is over the gel 1~2mm

PCR&AGE

1.Experiment material:genome DNA of arabidopsis , primer（flu、cop1、plf1、phyB、pHhl1、hhl1）
2.Experiment reagent:ddH2O, dNTP,Taq enzyme,Mg2+,10×Buffer
3.Experiment apparatus:DNA thermal cycler, elctrophoresis apparatus,elctrophoresis tank,ultraviolet
detector, table centrifuge, 0.5ml &1.5ml centrifuge tube, micropipet 4.experiment steps:
    (1)disposal the reaction system
        2μl buffer（including 15mM MgCl2）
        1μl dNTPs
        1.5μl DNA
        1.0μl forward primer
        1.0μl reverse primer
        0.2μl Taq enzyme
        13μl ddH2O
        (primer：flu,cop1,plf1,phyB,pHhl1,hhl1)
    (2)Set the PCR
        94℃×5min + （94℃×30s +62℃×35s + 72℃×40s）×40+ 72℃×5min + 4℃
    (3)PCR product detection & AGE
        ①making the agarose gel（see 2016.07.25 Morning)
        ②sample application：use micropipet to add 2μl DNA sample from experiment 1 to point template, and then add 1 μl buffer, mix ,then add it into the sample hole. Add 10 μl marker into one side the sample hole.
        ③electrophoresis:turn on the power,adjust the voltage. It can be observe after 30min.
        ④observation:put the gel on the ultraviolet detector, turn on the UV light, white nucleic acid strip can be seen. Based on the thickness and flosorescence intensity as well as the marker, we can estimate the concentration and molecular weight of DNA.
5.result:the strip is very dull, and there are many spots around the picture
6.analyze:
    (1) We didn’t add add the reagent accurately, which led to not finishing the PCR that makes a low DNA concentration
    (2) The dye wasn’t mixed with DNA completely, which makes the strip very dull
    (3)The DNA extracted from the plants wasn’t pure, cause the appear of impurities
    (4)For the gel was prepared a day ago, it may be polluted during the long time, which brought some impurities

the second time for PCR&AGE

1.experiment material and experiment apparatus is the as the first time(2016.07.26)
2.experiment reagent :primers was decreased to two types(plf1 and hhl)
3.experiment steps:
    (1)PCR:the same as the first time (2016.07.26)
    (2)sample application：use micropipet to add 2μl DNA sample from experiment 1 to point template, and then add 1 μl buffer, 0.5μl SYBY GREEN,mix ,wait for 10min,and then add it into the sample hole. Add 10 μl markertot o point template, 0.5μl SYBY GREEN,mix, wait for 5min, then add it into one side the sample hole.
    (3),(4)is the same as the steps in the first time
4.result:the brightness of strips are usual, but many strips’ length are less than 1000bp
5.analyze:
    (1)The primers may not be enough, they couldn’t combine with DNA perfectly, which led to the residue of primer,and didn’t get the target gene.
    (2)When extracting DNA, some parts of the DNA may be enzymolysised by some DNA helicase, so the DNA can’t combine with the primers.

the third time for PCR&AGE

1.experiment material and experiment apparatus is the as the first time(2016.07.26)
2.experiment reagent :primers was decreased to two types(plf1 and hhl)
3.experiment steps:
    (1)PCR: the react sysytem is the same as the first time (2016.07.26), but the react settings was changed into:94℃×5min + （94℃×30s +60℃×30s + 72℃×2min）×30+ 72℃×10min + 4℃
    (2)AGE: the same as the second time
4.result: the brightness and length of strips are very usual,but there are some obvious entrainments
5.Analyze:
    (1)PCR might occur non-specificity amplification, which produced many small fragments
    (2)The voltage of AGE might be too high, which damaged the aperture of agarose gel
    (3)We might add too much primers
    (4)We might didn’t add the dNTPs correctly, which led to the increasing of mispairing rate