Bacterial DNA extraction scheme

bacteria
DNA
extraction scheme: Gliland-negative bacteria, such as E.coli, generally have three options.
, boiling template method - mainly used for
PCR

1, inoculation monobacteria in LB or brain heart tablet, continuous drawing of lines, 37 degrees Celsius
culture
18-24 hours.
2, scrape 1 to 2 inoculated ring moss add 150 microliths of three steamed water, mix well, 100 degrees Celsius boiling for 10 minutes.
3, 12000 ropm centrifugal 10 minutes, take clear, -20 degrees Celsius to save spare.
is the easiest to operate and
requirements for
reagents are low. The disadvantage is that the purity is not high enough and may contain impurities such as RNA and protein. However, the PCR reaction template for general detection purposes is sufficient.
some people say that the amplification of 4kb or less fragments can be used to make templates in this way, the editor with such template PCR can be expanded to 3500bp fragments. Editor's advice: This method gets a short template save time, it is highly recommended to re-create once a month.
II, CTAB/NaCl method
1, inoculation monobacteria in 5 ml LB, 30 degrees C culture overnight,
2, take 1 ml seed culture fluid into 100 ml 2% LB, 37 degrees C, 220r/min culture 16 hours;
3, 5000r/min centrifuge for 10 minutes, discarded to the Qing.
4, after adding 10 ml TE centrifugal washing, dissolve the bacteria with 10 mlTE, mix well, -20 degrees C to save the spare.
5, take 3.5 ml of bacterial suspension, add 184 μl 10%
SDS
, mixed, added 37 μl10mg/ml
protease
K, mixed, 37c warmed for 1 hour
6, added 740μl5mol/LNaCl, added 512μl CTAB/NaCl, mixed well, 65C warmed for 10 minutes.
7, add the same volume of chloroform /isosterol, mix well, 10000r/min centrifugation for 5 minutes, retained on the clear.
8, the addition of the same volume of phenols: chloroform: isoquinol (25:24:1), mixed, 10000r/min centrifugation for 5 minutes, retained on the clear.
9, add 0.6 times isopropyl alcohol, mix well, 10000r/min centrifugation for 5 minutes, collect DNA precipitation, with 70% ethanol centrifugation washing DNA precipitation.
10, dissolved DNA with 1 ml of TE, and added a final concentration of 20 μg/mlRNaseA, which was stored at 4 degrees C.
CTAB/NaCl method extracts DNA with higher purity, fewer protein impurities, and long shelf life, and editors prefer to add the final concentration of 20 μg/ml RnaseA to step 5, so that there is no RnaseA contamination in the end. Each step is more detailed, and the resulting DNA can be used for South blot.
suggest that long-term preservation of DNA can be put at -20 degrees C, but to minimize repeated freezing and thawing, otherwise affect the quality of DNA.
, saline
1, 1.5 ml of diaste
2, 12000rpm 30 s
3, 200ul lysate (with CTAB/NaCl method), 37c, 1hr

4, 66ul 5M NaCL, well mixed, 12000rpm 10min
5, upper clear gun head taken to new tube
6, iso volume phenol fully mixed, 12000rpm 3min, repeatedly pumped Raised to protein-free layer
7, water layer, and other volume chloroform mix, 12000rpm 3min
8, upper clear to new tube, 2x volume pre-cooled waterless ethanol
9, -20C, 20min, 140 00rpm, 15min
10, 400ul 70% cold ethanol wash
11,
dry
, 100ul 20ug/ml RNaseA, 37C, 30min
12, 2 times the volume of waterless ethanol precipitation, 70% cold ethanol washing
13, dry, dissolved in 100ul TE
between method one and method two, CTAB although the removal of sugar effect is better, but CTAB itself is also toxic, so this method abandoned CTAB.
Grady-positive bacteria, due to the structure of the cell wall and negative bacteria have differences, lysis than negative bacteria slightly more troublesome, can take CTAB / NaCl method slightly modified, in the fourth step to add the final concentration of 2 mg / ml of lysomal enzymes, 37 degrees warm breeding 1h.
: It's best to cut off the tip of the gun in the
gene
group (cut it off quickly on the alcohol lamp to make it sleek). Lest the gun damage the genome! It's best to air dry when it's dry!
more sophisticated experiments, such as Southern, are highly recommended!