【交流】关于乳酸杆菌的电转化和质粒提取
丁香园论坛
2783
走过很多弯路,也做了许多无用功,现在终于有了一点经验,拿出来请大家指教
电转化protocol:
a.incubation in MRS (10mL)with 1% glucose for 6 hours till OD600=0.4-0.6
b.Pretreated with 10ng/mL Ampicillin for 60 min
c.Washing with washing buffer[5mM NaPO4 (pH 7.4),1mM MgCl2] two times, and then washing with electroporation buffer [0.9M sucrose,3mM MgCl2(pH 7.4)] one time, resuspended in electroporation buffer (150uL)
d.Add 1.5uL DNA in TE buffer(0.1-1ug) into 50 uL bacteria, mix, 5min on ice. Then add into 1mm gap cuvettee, electroporate with the following conditions:
R=400ohm,C=25uF,V=10kV-12.5kV/cm
e.After electropration, add 450uL MRS with 80mM MgCl2 into the cuvettee immediately. Transfer the bacteria in a eppendorf tube and incubate 3 h at 37C.
f.Plate 150uL bactedria on MRS plates containing 0.5ug/mL and 5ug/mL erythromycin for 24-48 h at 37C.
g.Pick clones.
References:
[1]http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12957947
[2]An improved method for the transformation of Lactobacillus strains using electroporation, Journal of Microbiological Methods 21 (1995) 97-109
质粒提取:
参考tzd等的经验(http://www.dxy.cn/bbs/thread/2120378),收集2ml菌液,先用含lysozyme的裂解液400ul[15％PEG1000，10mmol Tris.cl（pH8.0），10mg/ml溶菌酶]在37℃作用3小时,离心取沉淀后用普通的小量提取试剂盒就可以,我用的是BioDev的.
其中tzd是用了PEG2000,我用的是PEG1000.而我的lysozyme浓度较大,作用时间也比tzd的长,我懒得做优化了,如果谁有更好的办法请告诉我^_^
电转化protocol:
a.incubation in MRS (10mL)with 1% glucose for 6 hours till OD600=0.4-0.6
b.Pretreated with 10ng/mL Ampicillin for 60 min
c.Washing with washing buffer[5mM NaPO4 (pH 7.4),1mM MgCl2] two times, and then washing with electroporation buffer [0.9M sucrose,3mM MgCl2(pH 7.4)] one time, resuspended in electroporation buffer (150uL)
d.Add 1.5uL DNA in TE buffer(0.1-1ug) into 50 uL bacteria, mix, 5min on ice. Then add into 1mm gap cuvettee, electroporate with the following conditions:
R=400ohm,C=25uF,V=10kV-12.5kV/cm
e.After electropration, add 450uL MRS with 80mM MgCl2 into the cuvettee immediately. Transfer the bacteria in a eppendorf tube and incubate 3 h at 37C.
f.Plate 150uL bactedria on MRS plates containing 0.5ug/mL and 5ug/mL erythromycin for 24-48 h at 37C.
g.Pick clones.
References:
[1]http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12957947
[2]An improved method for the transformation of Lactobacillus strains using electroporation, Journal of Microbiological Methods 21 (1995) 97-109
质粒提取:
参考tzd等的经验(http://www.dxy.cn/bbs/thread/2120378),收集2ml菌液,先用含lysozyme的裂解液400ul[15％PEG1000，10mmol Tris.cl（pH8.0），10mg/ml溶菌酶]在37℃作用3小时,离心取沉淀后用普通的小量提取试剂盒就可以,我用的是BioDev的.
其中tzd是用了PEG2000,我用的是PEG1000.而我的lysozyme浓度较大,作用时间也比tzd的长,我懒得做优化了,如果谁有更好的办法请告诉我^_^
