I am from Mike Halen's, lab Department of Physiology and Biophysics, university of California, irv. Today I will show you how to use collagen mini fabricate to purify DNA from bacteria. I'm doing this as one step of a site directed mutagenesis experiment.
After I get plasmid, I will sequence the insert to verify that selected clones contain the desired mutations. Before we do the mini preparation, briefly introduce what we did yesterday. In order to grow bacteria, I need first inoculate five milliliter A LB, into individual tubes.
And as you can see, before we put bacteria into the tube, the median is clear. And then I use PERS to inoculate single colony from an airb plate and I switch tip every time so there's no contamination. And then I put tubes in a shaker and using 300 RPM to grow for at least an eight hours and normally we can do it overnight.
There are three important buffers from the collagen mini preparation kit. They are P one, P two, and N three. We normally put P one at four degree because it contains RNAs P two, we leave it at room temperature and for N three and we will pre it, it just before the experiment after eight hours.
As you can see, the median now is cloudy because bacteria, bacteria is growing inside and I'll see five microliter of the media onto another plate. So once I verify the clones, I can go back and directly to the bacteria and don't need to do another transformation. After we spin the median, you can see a nice bacteria pal at the bottom of the tube and we'll pull off the supat into bleach so there's no any contamination of the environment and the palate is still there.
Now I a the buffer P one and to the bacteria palates buffer. P one is resuspension buffer to resp bacteria. And now I'll pipee the solution up and down to where mix the bacteria with the buffer.
At this stage, you don't want to see any bacteria palette in the solution. Now I'll transfer the resus bender bacteria into a new appol tube. I always label my tubes so I can trick them and know which solution come from which clone.
Now I ate the buffer P two to each tube and buffer P two is a lysis buffer to lysate bacteria. After I the buffer P two, you can see the solution is still not clear. Now I are invert all the tubes for several times so the solution will mix well.
And now you can see the solution becomes clear. Now I a the buffer N three and to each tube buffer M three is neutralization buffer. Once I A the buffer N three, I immediately see the protein precipitation, but I'll still inverse them several times to mix the solution thoroughly.
After we mix the solution thoroughly. This is how it should look like after I aid in buffer and three. Now I'm going to use the sge to spin down the protein precipitation during the centrifugation for 10 minutes I assembled a Cal VAC six s.
I put all the pieces together in order and then connects them to a vacuum system. Now I aid the cal prep aid, the strap to the manifold. Today we'll only do one so I block or other slots.
So we have a good vacuum. As you can see, I labeled the strap according to my tubes and once I turn on the vacuum I have a good vacuum. Okay, after spinning the tube, you can see the protein pal at the bottom and DNA should still dissolve in the sup natant.
Now our transfer, the sup sup natant, which contains DNA to the cow prep strips I should mention, we always need to switch to tips and to prevent cross contamination. Now I switch on the vacuum. Now I switch off vacuum and aid buffer PB to wash the strip, which right now contains DNA in the filter.
Now I aid the solution PB to the strip to wash it and the strips still contains DNA in the filter. This time I don't need to switch the tips if I don't touch the strip. Now I a the buffer p to the strip in the same way as I did for the buffer pb.
And I'll do this for two times after I turn on the vacuum. As you can see, the P solution right now is going through the strip and we will leave the vacuum on for another five minutes to make sure the filter will be very dry After five minutes vacuum, take the strap out of the box and then wrap it with Kim wep. Hit it against the table to remove the residue P buffer.
And now you can see there's not many drop lid of P buffer and the DNA should still be in the filter. Here is the collecting micro tubes. I labeled it it according to the strip.
Now I open the car vac and and reassemble it with the collection micro tubes and make sure the strap match the micro tubes. This is how the manifold right now should look like. Here is a look of the vac manifold, which is prepared for eluting DNA from the strip to the micro tube.
The strip is on top and the tube is right under it, labeled in the same way. Now either sterile water to the strip without vacuum and let it stand for a minute. This time when vacuum is long, as you can see, water is going through the strip and get into the collecting micro tubes.
The water contains the DNA. I also leave the vacuum on for a while to make sure all the water will be collected. Here are the collection micro tubes with the DNA solution after the step.
Hi, I have just shown you how to use calcium mini prep kit to purify NA from bacteria. Now I are sequenced the insert to verify the right clones and I should mention using this method. The year of DNA is usually around the microgram level.
And good luck with your experiments. Thank you for watching.
