6203NATSCI Applications of Genetics in Health and Disease Practical 1

Today we are going to begin the procedure of using DNA sequencing to profile the bacteria present on your phones. To do this we will firstly swab the bacteria from your phone then we will extract DNA from the bacteria and then finally set up a PCR to amplify bacteria using the 16SrRNA gene (which is routinely used to identify bacteria to species).

You will work in pairs. So decide 1) whose phone you want to swab and 2) whose phone you want to put on agar to see what bacteria grow. The only purpose of putting the phone on LB agar is just to see what bacteria will grow and not for identification. I will take pictures of the bacteria colonies growing on the agar and put them on Canvas later in the week.

At any point if you don’t understand what is a spin column or Eppendorf or collection tube etc. I’ve put pictures at the back of this document.

  1. To swab bacteria of phone:


  1. Take your swab and dip it in the sterile water and swab the face of your phone for 2 minutes.
  2. Add 20 µl of PROTEINASE K and 180 µl DIGESTION SOLUTION (Buffer ATL) to a 1.5 ml Eppendorf tube.
  3. Swirl the swab in the solution in the 1.5 ml Eppendorf tube for 2 minutes.
  4. Cut off the swab tip (with scissors) and leave in the liquid.
  5. To extract DNA from your swab:
  6. Close the tube lid and incubate at 56⁰C for 10 mins on heating block/water bath.
  7. Remove swab tip, squeeze out as much liquid as possible and dispose of swab.
  8. Add 200 µl of LYSIS SOLUTION (Buffer AL). Mix thoroughly by vortexing to obtain a uniform suspension.
  9. Add 400 µl of 100% ETHANOL and mix by vortexing. Transfer the prepared lysate to a DNA purification Column (see pictures last page!) inserted in a collection tube. Centrifuge the column for 1 min at 8,000 rpm. IT IS IMPORTANT THAT WHEN YOU CENTRIFUGE YOU DO IT WITH ANOTHER GROUP AS THE CENTRIFUGE MUST BE CORRECTELY BALANCED!
  10. Discard the collection tube containing the flow-through solution. Place the DNA Purification Column into a new 2 ml collection tube.
  11. Add 500 µl of WASH BUFFER 1 (AW1) to the DNA Purification Column. Centrifuge for 1 min at 10,000 rpm. Discard the flow-through and place the purification column back into the collection tube.
  12. Add 500 µl of WASH BUFFER 2 (AW2) to the DNA Purification Column. Centrifuge for 3 mins at maximum speed (13,000 rpm).
  13. Discard the collection tube containing the flow-through solution and transfer the DNA Purification Column to a sterile 1.5 ml Eppendorf tube and chop lid off with scissors.
  14. Add 50 µl of ELUTION BUFFER (Buffer AE) to the center of the DNA Purification Column membrane to elute genomic DNA. Incubate for 2 min at room temperature and centrifuge for 1 min at 10,000 rpm.
  15. Your bacterial DNA is now at the bottom of the 1.5 ml Eppendorf tube so you can throw away the purification column and progress to PCR.
  16. To set up PCR of bacterial 16SrRNA gene

We are going to set up two PCR reactions. One will contain the bacterial DNA you just extracted and the other will contain no DNA, just water (negative control), and will check whether you have contaminated your reagents with bacteria during the DNA extraction process!

The first thing to do is take 2 PCR tubes. They have already been labelled with a number (that’s your group) and on the side one tube is called “+ve” and the other “-ve” corresponding to the tube that has bacterial DNA (+ve) in it and the other which has just water (-ve).

To each of the tubes you need to add the following:

PCR Master Mix:                                                         13 µl

Forward Primer called 27F:                                        2 µl

Reverse Primer called 1492R:                                    2 µl

DNA that you extracted (or water if –ve control):     2 µl

Water:                                                                         6 µl

PLEASE REMEMBER: The negative control will have all the reagents added but NO DNA and 2 µl of water added instead!

FYI: Forward primer contains the DNA sequence: 5’-AGAGTTTGATCMTGGCTCAG-3’

Reverse primer contains the DNA sequence: 5’-TACGGYTACCTTGTTACGACTT-3’, both are at concentration of 10 µM.

Once you have added the ingredients to each tube please put the lid on each, label with your initials and give them to Robbie. This is the end of Practical 1 and (fingers crossed!) you have managed to successfully extract DNA from your bacteria!

The next stage is to run the PCR which I will carry out. The conditions are as follows (if interested):

3 mins at 95⁰C

15 seconds at 95⁰C

35 cycles

30 seconds at 55⁰C

1.5 mins at 72⁰C

8 mins at 72⁰C

Finish at 16⁰C

Has my PCR worked? In order to understand this we have to run a gel. I will do this before the next practical and put the images up on canvas. If it has worked then we will proceed to the next stage which is cloning the PCR product and then heat shocking the bacteria into E. coli (see Prac 2).