分子生物學(xué)實(shí)驗(yàn)技能操作ek lic cloning and expression

1、EK/LIC cloning and expression1. T4 DNA polymerase treatment of target insert 1) Make the EK/LIC GeneGenesPCR (l) (0.1pmol)ddH2O (l)10*T4 DNA Polymerase buffer(l)100mM dATP(l)BSA(l)T4DNA Polymerase (2.5U/ l, l)Calkro_20080.53.050.50.250.50.2Calkro_23860. 53.050.50.250.50.2加樣量(l)Final concentrationGen

2、e EK/LIC DNAa (a3.55)0.1 pmolddH2O3.55-a10*T4 DNA Polymerase buffer0.51*dATP (100 mM)0.255 mMBSA (0.1%)0.50.01%T4 DNA Polymerase (5 U/l)0.20.2 UTotal52) Start reaction by adding 0.2l of 2.5U/ l T4DNA Polymerase3) Incubate at 37 Celcius for 30min 4) Inactivate enzyme by incubating at 75 Celcius for 2

3、0min2. T4 DNA polymerase treatment of EK/LIC pET-28b Vector1) Make the Vector:Genes(pet28:300 ng/uL): PCR (l) ddH2O (l)10*T4 DNA Polymerase buffer(l)100mM dTTP(l)bsaT4DNA Polymerase (2.5U/ l, l)12931.231.84310.410.62.3 uL is 0.1 pmol, so the amount in the mixture is around 0. 173 pmol加樣量(l)Final con

4、centrationpET-28b EK/LIC DNAb (b3.5)0.03 pmolddH2O3.5-b10*T4 DNA Polymerase buffer0.51*dTTP (100 mM)0.24 mMBSA (0.1%)0.50.01%T4 DNA Polymerase (5 U/l)0.30.3 UTotal52) Start reaction by adding 1.80.3 uL of 2.5U/ l T4 DNA Polymerase3) Incubate at 37 Celcius for 30min 4) Inactivate enzyme by incubating

5、 at 75 Celcius for 20min3 Annealing the Vector and Ek/LIC Insert 1) Make the componentsuLFinal concentration-Ek/LIC vector-pet2810.03 pmolgene320.3 pmolMixed gently, and centrifuge for 10 second.2) Incubate at 22 Celcius (room temperature) for 15 min3) Add 0.5l of 25mM EDTA, mix gently, then short-t

6、erm centrifuge4) Incubate at 22Celcius for 5min4 TransformationAdd 1 uL annealed sample to 100 uL competent cells TOP10.Leave on ice for 30 min;Heat shock 42degree, indicated as 41 degree) for ?60s; Leave on ice for 2 min, then add 200uL 500 uL LB medium and culture at 37degree for 1 hour;5000 rpm c

7、entrifuge for 3 min, discard some supernatant.Spread on solid LB meidium with kanamycin, and cultured at 37 degree. Note: How to calculate how much volume PCR production is 0.1 pmol. A: DNA concentration (ng/L)B: Molecular weight(Da)0.1 pmol (L) b (0.15 pmol)a (0.5 pmol)ABB*10-4/AB*10-4/A *1.5B*10-4

8、/A *5Pet28b 3317034.20 Da 5368 bpTimePet28b Molecular weight(Da)pET28b EK/LIC DNA concentration (ng/L)b (0.15 pmol)(b3.5 L)2013-10-143317034.20303.001.6432013-10-163317034.20134.003.713TimeGeneMolecular weight(Da)DNA concentration (ng/L)a (0.5 pmol)(a3.55 L)菌落個(gè)數(shù)（供參考）2013-10-16Calla_199329821.35 190.

9、500.07811 (1 uL轉(zhuǎn)感受態(tài)) /47 (3 uL轉(zhuǎn)感受態(tài))Calla_001855840.98 84.000.33280Calla_052157874.99 106.000.27311Calla_178178878.82 74.500.53054Calla_125980343.31 83.000.48413Calla_238595350.86 之前DNA分子量被誤算為蛋白分子量，所以實(shí)驗(yàn)結(jié)果僅供參考。之前DNA分子量被誤算為蛋白分子量，所以實(shí)驗(yàn)結(jié)果僅供參考。205.500.2328Calla_1993496767.80190.501.304Calla_0018924906.3584.005.505Calla_0521937858.24106.004.424Calla_178112