PROJECT LAB BOOK
Welcome to the Macquarie University project lab book page!
Here you will find a day-by-day account of our triumphs and failures.
A day-by-day progress for Agrobacterium Tumefaciens Bacteriophytochrome
20th August 2010
Genomic DNA extraction
- The first primer pair is (AT-BHO-F) with (AT-BHO-R) [this will insert the bacteriophytchrome gene BEFORE the heme oxygenase gene in the operon]
- The second primer pair is (AT-AHO-F) with (AT-AHO-R) [this will insert the bacteriophytochrome gene AFTER the heme oxygenase gene in the operon]
A technique called gradient PCR will be used here. This PCR includes different annealing temperatures so that the optimum annealing temperature for the primers can be determined.
This should also result in reduced non-specific binding that was observed in the previous PCR result
The reaction mastermix for the PCR was set up as per the following recipe (per sample):
Mastermix: |
Amount per sample (ul) |
Gibco H2O |
13.75 |
10x Buffer |
2.00 |
Polymerase enzyme |
0.25 |
dNTP |
1.00 |
Fwd primer |
1.00 |
Rvs primer |
1.00 |
Genomic DNA |
1.00 |
Total |
20.00 |
The PCR program was set up as per the following:
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 60˚C for 30 seconds
- 72˚C for 2 minutes & 30 seconds
(This was repeated for another 25 cycles)
- 72˚C for 10 minutes
- 4˚C to end.
The PCR products were run on a GelRed post-stained 2% agarose gel using a 1kb ladder for visualization
The products were run on a GelRed post-stained 2% agarose gel for visualization
No products were observed
Experimental Design – Primer combinations and annealing temperatures:
Fwd Primer |
Rvs primer |
Temp 1 (Degrees Celsius) |
Temp 2 (Degrees Celsius) |
Temp 3 (Degrees Celsius) |
Temp 4 (Degrees Celsius) |
Temp 5 (Degrees Celsius) |
(AT-BHO-F) |
(AT-BHO-R) |
57.1 |
58.7 |
60.6 |
63.4 |
64.8 |
(AT-AHO-F) |
(AT-AHO-R) |
57.1 |
58.7 |
60.6 |
63.4 |
64.8 |
Figure 4. PCR Optimisation (gradient PCR) results
Figure 4. No product amplification is seen in any lanes. The anticipated product is approximately 1.6 to 3.0kb. The gel had been over run and at the very bottom some bands can be seen but as these are so small they are possibly dimers that are less than 300bp.
No products were observed. The anticipated product size was between 1.6kb and 3kb. The gel was over run but the only products that were over run were probably primer dimers, which are less than 300bp in size. = FAIL!
7th September 2010
PCR Optimization (using Gradient PCR)
The PCR run on 3rd September wasn’t successful so this was repeated
This time however, only the PCR product was used as a template in two different dilutions (1:100 and 1:200)
Additionally, two different annealing temperatures were used: 60 and 65C
The primer pairs used in the previous PCR were also used again for the insertion of the bacteriophytochrome gene and heme oxygenase in different orientations in the operon
The reaction mastermix for the PCR was set up as per the following recipe (per sample):
Mastermix: |
Amount per sample (ul) |
Gibco H2O |
13.75 |
10x Buffer |
2.00 |
Polymerase enzyme |
0.25 |
dNTP |
1.00 |
Fwd primer |
1.00 |
Rvs primer |
1.00 |
Genomic DNA |
1.00 |
Total |
20.00 |
The PCR program was set up as per the following:
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 60˚C for 30 seconds
- 72˚C for 2 minutes & 30 seconds
(This was repeated for another 25 cycles)
- 72˚C for 10 minutes
- 4˚C to end.
The PCR products were run on a GelRed post-stained 2% agarose gel using a 1kb ladder for visualization
There was no amplification observed in any of the lanes so the picture of this gel is not included = FAIL!
Experimental Design – Primer combinations and annealing temperatures:
DNA template |
Dilution |
Fwd primer |
Rvs primer |
Annealing temp (Degrees Celsius) |
PCR product (from DNA2.2) |
1:100 |
(AT-BHO-F) |
(AT-BHO-R) |
60 |
PCR product (from DNA2.2) |
1:200 |
(AT-BHO-F) |
(AT-BHO-R) |
60 |
PCR product (from DNA2.2) |
1:100 |
(AT-AHO-F) |
(AT-AHO-R) |
60 |
PCR product (from DNA2.2) |
1:200 |
(AT-AHO-F) |
(AT-AHO-R) |
60 |
PCR product (from DNA2.2) |
1:100 |
(AT-BHO-F) |
(AT-BHO-R) |
65 |
PCR product (from DNA2.2) |
1:200 |
(AT-BHO-F) |
(AT-BHO-R) |
65 |
PCR product (from DNA2.2) |
1:100 |
(AT-AHO-F) |
(AT-AHO-R) |
65 |
PCR product (from DNA2.2) |
1:200 |
(AT-AHO-F) |
(AT-AHO-R) |
65 |
10th September 2010
PCR Optimization (repeated)
As we were having trouble with one of the primers annealing we attempted a PCR technique that required a different PCR program to be set
The (AT-AHO-F)– (AT-AHO-R)primer pair were run on the normal PCR program being used for all other PCR’s as these primers were annealing properly
The primer pair that we were having difficulty with ((AT-BHO-F)-(AT-BHO-R)) required a special PCR program. This would allow for the (AT-BHO-R)primer to anneal to the template first for single stranded amplification and then the (AT-BHO-F)primer to bind later.
The reaction mastermix for the PCR was set up as per the following recipe (per sample):
Mastermix: |
Amount per sample (ul) |
Gibco H2O |
13.75 |
10x Buffer |
2.00 |
Polymerase enzyme |
0.25 |
dNTP |
1.00 |
Fwd primer |
1.00 |
Rvs primer |
1.00 |
Genomic DNA |
1.00 |
Total |
20.00 |
The first PCR program was set up as per the following:
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 60˚C for 30 seconds
- 72˚C for 2 minutes & 30 seconds
(This was repeated for another 35 cycles)
- 72˚C for 10 minutes
- 4˚C to end.
The second PCR program was set up as per the following (this was for the amplification of product using the (AT-FWD-RBS) and (AT-AHO-R) primer pair):
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 40˚C for 30 seconds
- 72˚C for 2 minutes & 30 seconds
(This was repeated for another 35 cycles)
- 72˚C for 10 minutes
- 4˚C to end.
- The second PCR program was set up as per the following. There were two parts to this PCR to allow for the (AT-BHO-F) primer to anneal properly after initial annealing of the (AT-BHO-R) primer.
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 40˚C for 30 seconds
- 72˚C for 2 minutes and 30 seconds
(This was repeated for another 4 cycles)
- Now we can add the (AT-BHO-F) primer to the reaction.
- 4˚C for 5 minutes
- 94˚C for 30 seconds
- 60˚C for 30 seconds
- 72˚C for 2 minutes and 30 seconds
(This was repeated for another 31 cycles)
- 72˚C for 10 minutes
- 4˚C to end
- The PCR products were run on a GelRed post-stained 2% agarose gel using a 1kb ladder for visualization
Experimental Design – Primer combinations and annealing temperatures:
Template |
Primer pair |
Annealing temp (degrees Celsius) |
Number of cycles |
(AT-FWD-RBS)-(AT-RVS-1)PCR product |
(AT-BHO-F)-(AT-BHO-R) |
40, then 60 |
4, then 35 |
(AT-FWD-RBS)-(AT-AHO-R)PCR product |
(AT-AHO-F)-(AT-AHO-R) |
60 |
35 |
(AT-FWD-RBS)-(AT-RVS-1)PCR product (1:10 dilution) |
(AT-BHO-F)-(AT-BHO-R) |
40, then 60 |
4 then 35 |
(AT-FWD-RBS)-(AT-AHO-R)PCR product (1:10 dilution) |
(AT-AHO-F)-(AT-AHO-R) |
60 |
35 |
Figure 5. PCR optimization (with ssPCR amplification for (AT-BHO-F)primer)
Figure 5. GelRed post-stained 2% agarose gel of optimized PCR using primer pairs for insertion of the heme oxygenase gene and bacteriophytochrome gene. In lanes 1 and 6 there is a 1kb ladder. In lane 2 there is the (AT-FWD-RBS)-(AT-RVS-1)PCR product amplified with (AT-BHO-F)-(AT-BHO-R)primer pair on the special PCR program allowing for the (AT-BHO-R)primer to bind first. In lane 3 there is the (AT-FWD-RBS)-(AT-AHO-R)PCR product amplified with (AT-AHO-F)-(AT-AHO-R)primer pair on the normal PCR program. In lane 4 there is the (AT-FWD-RBS)-(AT-RVS-1)PCR product diluted 1:10 amplified with (AT-BHO-F)-(AT-BHO-R)primer pair on the special PCR program allowing for the (AT-BHO-R)primer to bind first. In lane 5 there is the (AT-FWD-RBS)-(AT-AHO-R) PCR product diluted 1:10 amplified with (AT-AHO-F)-(AT-AHO-R)primer pair on the normal PCR program. There is a product band seen in lane 3 – SUCCESS! This is the product with the bacteriophytochrome gene inserted AFTER the heme oxygenase gene.
Discussion of gradient PCR:
The gradient PCR was used because the (AT-BHO-R) primer that was ordered was 12 base pairs shorter than what was supposed to be ordered. The (AT-BHO-R)primer was supposed to include the whole reverse primer sequence (18 base pairs) as well as an additional sequence for the HO site. This caused an issue with this primer annealing in previous PCR reactions. The gradient PCR will allow for this primer to anneal prior to any other primer annealing increasing the chance of annealing.