15-16 April 2017 workshop



We had 11 kids from 8 to 15 years old joining the 2 day workshop. In the workshop, they collected 33 plant samples in total, and then using some basic molecular biology lab skills including DNA extraction, PCR and gel electrophoresis to generate the barcode of these plant samples.

Experiment part Day Time  Activity   
 I. Collect samples and document
 1

30 min

75 min
Pre-lab:

Lab: 
 Introduce what is DNA Barcoding and iNaturalist
Field trip at Lei Yue Mun,Collect tissue or processed material
 II. Isolate DNA from plant samples
 1  45 min


 120 min
 Pre-lab:



Lab:
Lecture on DNA extraction
Aliquot reagents
Set up student stations
Teach how to use micropipette
Isolate DNA
 III. Amplify DNA barcode by PCR
 2
 15 min
 


10 min
120 min
Pre-lab:




Lab:

Lecture on PCR
Aliquot ddH2O, primers and Taq polymerase
Set up PCR profile
Set up student stations
Mixing reagents for PCR reaction
Amplify DNA in termal cycler
 IV. Analyze PCR products by Gel electrophoresis
 2  30 min




60 min
Pre-lab:




Lab: 
 Lecture on gel electrophoresis
prepare 1X TAE buffer
Set up student stations (agarose powder, casting tray, weight balance, DNA stain)
Cast gels
Load DNA samples into gels
Run gel electrophoresis
Photograph gels


I. Collect samples and document:
We use iNaturalist to document our collections. Photos, geolocation and notes are added to the DIYBIOHK Barcode Hong Kong Project.
Note: 15/04 #1-33
Number of samples: 33


II. Isolate DNA from plant samples
We used the DNeasy Plant kit from qiagen. Each students worked on 3 samples.


III. Amplify DNA barcode by PCR
4ul DNA template was added to each pcr tube
amplify rbcL gene

Reaction mixture:
 

Tube

Template DNA

4ul

Primer 1

(10umole rbcLa f)

2ul

Primer 2

(10umole rbcLa rev)

2ul

Taq Master Mix

25ul

ddH2O

17ul

Total

50ul


PCR profile:

94℃

5 min

 

94℃

30 sec

35 cycles

54℃

30 sec

72℃

60 sec

72℃

7min

 

4℃

Forever

 

Since the limited place in the PCR machine, we only pcr 20 samples at the workshop.  So we still need to do 2nd bench of PCR to finish all 33 samples.

IV. Analyze PCR products by Gel electrophoresis


Gel 1
Well   1 10  11 
Sample  ladder #2   #3  #4 #5  #8  #9  #10  #11  #14  #16
presence of DNA band
(x = presence)
 x  x  x  x  x  x    x  x  x  

Gel 2
Well   1 10  11 
Sample  ladder # 17  #20  #21 #23  #24  #27  #29  #30  #31  #32
presence of DNA band
(x = presence)
 x  x  x  x
   x        

  Fig. 1 Gel photo of PCR products. 7ul pcr products was added in each well. The DNA barcode (rbcL) gene is ~600-700bp.

The DNA bands are too thick, it is recommended to load less volume (5ul) sample to the well. The DNA ladder is blurry, may be the gel electrophoresis was running too fast or the DNA ladder was degraded.


Next step:
- Send the successful pcr products (barcode) to do DNA sequencing.
- Develop a more economic DNA extraction method, with reference to the Barcoding 101 protocol.

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