Team:Macquarie Australia/Notebook

A. tumefaciens genomic DNA extraction agarose results:

Figure 1. Results of A. tumefaciens genomic DNA extraction

Figure 1. GelRed stained 1% agarose gel of genomic DNA extraction from A. tumefaciens.

In lane 1 there is a 1kb ladder. In lane 2 is the DNA1.1 flow through, lane 3 is the DNA1.2 flow through, lane 4 is the DNA2.1 flow through and lane 5 is the DNA2.2 flowthrough. All four samples show a smear that is indicative of genomic DNA. The extraction has been successful.

Nanodrop absorbance readings:

Overall, the Nanodrop readings show that we have obtained good DNA concentration with minimal protein contamination

= SUCCESS!

Fwd and Rvs primers for amplification of the full length A. tumefaciens BphP gene:

Fwd and Rvs primers for amplification of the full length A. tumefaciens BphP gene for insertion in the operon BEFORE the HO gene:

Fwd and Rvs primers for amplification of the full length A. tumefaciens BphP gene for insertion in the operon AFTER the HO gene in the operon:

Fwd and Rvs primers for amplification of the full length A. tumefaciens BphP gene for insertion in the operon AFTER the HO gene as well as the addition of a ribosome binding site or Shine Delgano sequence:

Initial PCR

The PCR program was set up as per the following:

1. 94˚C for 2 minutes
2. 94˚C for 30 seconds
3. 60˚C for 30 seconds
4. 72˚C for 2 minutes & 30 seconds

(This was repeated for another 25 cycles)

5. 72˚C for 10 minutes
6. 4˚C to end.

Experimental Design – Primer combinations:

Figure 2. Results of the initial PCR

In lanes 1 and 11 there is a 1kb ladder. In lane 2 is the DNA1.2 template with (AT-FWD-1) and (AT-RVS-1)primer pair.

In lane 3 is the DNA2.2 template with (AT-FWD-1) and (AT-RVS-1)primer pair. In lane 4 is the DNA1.2 template with (AT-BHO-F) and (AT-RVS-1)primer pair. In lane 5 is the DNA2.2 template with the (AT-BHO-F) and (AT-RVS-1)primer pair. In lanes 6 and 7 there is nothing loaded as the wells were damaged. In lane 8 there is the DNA1.2 template with (AT-FWD-RBS) and (AT-RVS-1)primer pair. In lane 9 there is the DNA2.2 template with the (AT-FWD-RBS)and (AT-RVS-1)primer pair and in lane 10 there is the ssH2O negative control. A product is seen in lane 9 – this is the DNA2.2 template with the (AT-FWD-RBS)and (AT-RVS-1)primers! This means that we have a A. tumefaciens bacteriophytochrome product with a ribosome binding sight inserted.

The PCR program was set up as per the following:

1. 94˚C for 2 minutes
2. 94˚C for 30 seconds
3. 60˚C for 30 seconds
4. 72˚C for 2 minutes & 30 seconds

(This was repeated for another 25 cycles)

5. 72˚C for 10 minutes
6. 4˚C to end.

Experimental Design – Primer combinations:

Figure 3. PCR optimization results

• 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]

The PCR program was set up as per the following:

1. 94˚C for 2 minutes
2. 94˚C for 30 seconds
3. 60˚C for 30 seconds
4. 72˚C for 2 minutes & 30 seconds

(This was repeated for another 25 cycles)

5. 72˚C for 10 minutes
6. 4˚C to end.

Experimental Design – Primer combinations and annealing temperatures:

Figure 4. PCR Optimisation (gradient PCR) results

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!

The PCR program was set up as per the following:

1. 94˚C for 2 minutes
2. 94˚C for 30 seconds
3. 60˚C for 30 seconds
4. 72˚C for 2 minutes & 30 seconds

(This was repeated for another 25 cycles)

5. 72˚C for 10 minutes
6. 4˚C to end.

Experimental Design – Primer combinations and annealing temperatures:

The first PCR program was set up as per the following:

1. 94˚C for 2 minutes
2. 94˚C for 30 seconds
3. 60˚C for 30 seconds
4. 72˚C for 2 minutes & 30 seconds

(This was repeated for another 35 cycles)

5. 72˚C for 10 minutes
6. 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):

1. 94˚C for 2 minutes
2. 94˚C for 30 seconds
3. 40˚C for 30 seconds
4. 72˚C for 2 minutes & 30 seconds

(This was repeated for another 35 cycles)

5. 72˚C for 10 minutes
6. 4˚C to end.
7. 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.
8. 94˚C for 2 minutes
9. 94˚C for 30 seconds
10. 40˚C for 30 seconds
11. 72˚C for 2 minutes and 30 seconds

(This was repeated for another 4 cycles)

12. Now we can add the (AT-BHO-F) primer to the reaction.
13. 4˚C for 5 minutes
14. 94˚C for 30 seconds
15. 60˚C for 30 seconds
16. 72˚C for 2 minutes and 30 seconds

(This was repeated for another 31 cycles)

17. 72˚C for 10 minutes
18. 4˚C to end
19. 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.

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