It is very easy,

To an Eppendorf tube add 20 ul of water (sterile of course), than use a tip for colony scratching from a Petri dish, put it into the Eppendorf, dissolve it by shaking, close the eppendorf and boil it for 5 min. Be careful, during boiling the eppendorf should be in vertical position,  because sometimes it is opening during boiling and you do not want to water gets inside. Just close it and boil till 5 min. Sometimes I am adding PrepMan, it is bounding all PCR inhibitors, if you do not have it it is also okay. Just remember that at the first PCR using such a template you have to add app. 5ul DNA as a template and sometimes you need a second PCR for re-amplification to see the results on a gel.

If you have some problems or additional question just write.

Best,

Tomasz

It is very easy,

To an Eppendorf tube add 20 ul of water (sterile of course), than use a tip for colony scratching from a Petri dish, put it into the Eppendorf, dissolve it by shaking, close the eppendorf and boil it for 5 min. Be careful, during boiling the eppendorf should be in vertical position,  because sometimes it is opening during boiling and you do not want to water gets inside. Just close it and boil till 5 min. Sometimes I am adding PrepMan, it is bounding all PCR inhibitors, if you do not have it it is also okay. Just remember that at the first PCR using such a template you have to add app. 5ul DNA as a template and sometimes you need a second PCR for re-amplification to see the results on a gel.

If you have some problems or additional question just write.

Best,

Tomasz

Tomasz Łukasz Cłapa thanks alot

Two to three colonies are suspended in TE buffer and subjected to centrifugation

at 1000 rpm for 3 minutes at 4°C. The suspension was then boiled at 100 °C

for 10 minutes.

Please try to look for this article regarding the method

Ivanov, I.G. and D.R. Bachvarov. 1987. Determination of plasmid copy

number by the “boiling” method. Analytical Biochemistry 165(1): 137-

141.

Search for 'Thermal Lysis' and you should get a number of protocols that will require optimising, depending on the equipment available and your specific requirements. Generally, if the bacteria are in suspension, you must remove the cells from the growth media by centrifugation, and then resuspend the pelletted cells in TE in a screw cap tube. If colonies are on agar plates simply remove one colony and resuspend in TE. The volume of TE used depends how concentrated/dilute you want your DNA to be. Then, place the tube in a boiling water bath for 8 minutes. Remove the tube from the water bath and centrifuge at ~13k rpm for 5 minutes. Without disturbing the pellet, remove the supernatant and transfer to a clean (irradiated) tube ready for analysis.

are those protocols useful in gram + bacteria? 

does it work for G+ bateria?

I work Listeria extraction with this method. It is usefull and very good. Especially in bacterial culture...

Iused boiling method for Extraction of Clostridium perfringens DNA and the steps were:

Five to ten colonies of C. perfringens from blood agar plates suspended in 200ul distilled water in eppendorf tube using micropipettes. The samples were boiled at 100°C for 20 minutes, snapped cooled on ice for 5 minutes and centrifuged at 13.000 revolutions per minute for 3 minutes. The supernatant was transferred to clean tubes and stored at -20°C.

Can I use this method on my fungi samples?

@Hossein Jamil, Can you please enlighten me regarding the purpose of keeping the cells at -20 after boiling?

Thank you in advance.

The rapid boiling method performed according to Diana, J., C. Pui, et al. (2012). with slight modified. In brief, 0.5, 1 and 1.5 ml of each overnight bacterial culture was centrifuged at 13,000 rpm for 5 min at 4°C and the supernatant was carefully removed and the pellet was suspended in 500 µl of sterile distilled water. The sample was then boiling for 15 min in a water bath and immediately cooled at -20°C for 10 min prior to centrifugation at 13,000 rpm for 5 min at 4°C, and supernatant containing genomic DNA transfer in new tube and it was used for subsequent PCR amplification.

The rapid boiling method performed according to Diana, J., C. Pui, et al. (2012). with slight modified. In brief, 0.5, 1 and 1.5 ml of each overnight bacterial culture was centrifuged at 13,000 rpm for 5 min at 4°C and the supernatant was carefully removed and the pellet was suspended in 500 µl of sterile distilled water.

Interesting, i tried this method with protozoan parasites. It wasnt so consistent in comparison to commercial kits. I think more still remains to be done in streamling the temperature for different pathogens.