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E. coli cells can be disrupted in an alkaline solutioncontaining detergent. The lysate contains enough DNA to be detectedin a single lane of an agarose gel provided that the plasmid havepUC-derived replication origin. The following protocol is modifiedfrom that described in the first version of 'Molecular Cloning'(T. Maniatis, E. F. Frisch and J. Sambrook, Cold Spring HarborLaboratory Press, 1982). (NOTE: I seldom use this procedure these days,since PCR-based method described hereis more reliable.)

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1. Grow bacterial colonies to a large size (2-3 mm) on an agar medium contining an appropriate antibiotic.

2. Using a sterile toothpick, transfer a small quantity of the colony to a master plate. Transfer the remainder of the colony to a microfuge tube containing 20 microliters of 50 mM NaOH, 0.5% SDS, 5 mM EDTA (cracking buffer).

As illustrated in the animated picture of PCR, three major steps are involved in a PCR.These three steps are repeated for 30 or 40 cycles. The cycles are done on an automated cycler, a device which rapidly heats and cools the test tubes containing the reaction mixture. Cracked.to is a community forum that suits basically everyone. We provide checking tutorials, tools, leaks, marketplace and many more stuff! You can also learn many things here, meet new friends and have a lot of fun! To crack an 8 digit password with 95 different available characters (26.2 letters (upper and lower), 10 digits, 33 special characters) there are 95 8 = 6.9063e+15 combinations To crack it in one week you would need to be testing 95 8 / (7. 24. 60. 60) = 10,969,253,162 passwords PER SECOND.

3. Incubate the tube at 55 C for 30 min.

4. Vortex vigorously for 1 min.*

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5. Add an appropriate amount of loading buffer**. Load the contents onto an agarose gel. As a control, load the plasmid vector without insert on one lane. (Note: It may be very difficult to apply the cracking mixture into the well because of its high viscosity. Loading the mixtures into empty wells rather than the wells filled with buffer and pouring electrophoresis buffer thereafter may give better result.)

6. After electrophoresis, stain the gel by soaking it for 30 minutes in a solution of ethidium bromide (0.5 microgram/ml in electrophoresis buffer).

7. Under UV-illuminator, plasmid DNA should be visible between E. coli genomic DNA (20-30 kb) and low molecular weight RNAs.

* At this step, long genomic DNA is cut into smaller pieces of about 20-30 kb. Although the original protocol in 'Molecular Cloning' does not contain this step, vigorous vortexing is necessary since long genomic DNA in the lysate is troublesome in loading the sample onto the agarose gel.
** Add the loading buffer just before electrophoresis, since bromophenol blue is rapidly degraded in the alkaline solution.

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