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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

A. tumefaciens genomic DNA extracted using the Invitrogen PureLinkTM Genomic DNA Purification kit as per the manufacturer’s protocols.

The extractions were run on a GelRed stained 1% agarose gel and photo taken for visualization (see below).

A NanoDrop spectrophotometer reading was also recorded to check the quality of the extracted genomic DNA.

The extraction was successful for all A. tumefaciens cell lysate samples (labeled DNA1.1, DNA1.2, DNA2.1, DNA2.2 (See figure below)

A. tumefaciens genomic DNA extraction agarose results:

All four DNA samples show a smear of gDNA. Because the samples were treated with RNase there is no band indicative of RNA visible = SUCCESS!

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:

Genomic DNA sample	260/280 OD ratio	Concentration (ng/mL)
DNA1.1	1.88	351.5
DNA1.2	1.98	203.1
DNA2.1	2.14	738.9
DNA2.2	2.16	709.7

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

= SUCCESS!

27th August 2010

Primer design

Various primers were designed manually and using Primer 3 Software package for PCR amplification.

The primers were ordered and supplied through IDT.

There was an array of various primers ordered for amplification of different products. The details of the primers are described below.

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

Primer name	Primer Sequence
(AT-FWD-1)	5’-ATG AGT TCA CAT ACG CCG-3’
(AT-RVS-1)	5’-TCA GGC AAT TTT TTC CTC-3’

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

Primer name	Primer Sequence
(AT-BHO-F)	5’- AAG GAG ATA TAC ATA TGA TGA GTT CAC ATA CGC CG – 3’
(AT-BHO-R)	5’- AAG TTG ACA CTC ATA TGA GCC CTC CTT TCA GGC – 3’

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:

Primer name	Primer Sequence
(AT-AHO-F)	5’- CCG AAG GCT AGG ATC CAG GAG GGC TGC TAT GAG – 3’
(AT-AHO-R)	5’- GTT AGC CGG ATC CTC AGG CAA TTT TTT CCT – 3’

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:

Primer name	Primer Sequence
(AT-FWD-RBS)	5’- AGG AGG GCT ATG AGT TCA CAT ACG CCG -3’

Initial PCR

The reaction mastermix for the initial 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.

Different combinations of the primers were used for the PCR reaction (see below ‘Experimental Design’ section following)

Not all possible primer combinations were used due to limitations on the amount of polymerase enzyme available to us

The PCR products were run on a 1.2% GelRed post-stained agarose gel for visualisation (see picture of gel below).

Experimental Design – Primer combinations:

Template DNA	Fwd Primer	Rvs primer
DNA1.2	(AT-FWD-1)	(AT-RVS-1)
DNA2.2	(AT-FWD-1)	(AT-RVS-1)
DNA1.2	(AT-BHO-F)	(AT-RVS-1)
DNA2.2	(AT-BHO-F)	(AT-RVS-1)
DNA1.2	(AT-FWD-RBS)	(AT-RVS-1)
DNA2.2	(AT-FWD-RBS)	(AT-RVS-1)
SSH20 (negative control)	(AT-FWD-1)	(AT-RVS-1)

A band was seen in lane 9. This lane was using the primer and DNA template combinations of DNA2.2, (AT-BHO-F) and (AT-RVS-1) primers

= SUCCESS!!!

A PCR product is seen in lane 9 – this is the DNA2.2 template amplified with the (AT-FWD-RBS) and (AT-RVS-1) primers

= SUCCESS!!

Figure 2. Results of the initial PCR

Figure 2. GelRed post-stained 1.2% agarose gel of initial PCR of A. tumefaciens using various primers.

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.

30th August 2010
PCR Optimization

Now that we have a product obtained by the initial PCR it is time to optimize the PCR conditions using the successful DNA2.2 PCR product obtained from last week and the original DNA2.2 template

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.

Again, different combinations of the primers were used for the PCR reaction (see below ‘Experimental Design’ section following)

Now that we don’t have a limited enzyme supply, even more primer combinations can be used!!

The PCR products were run on a GelRed post-stained 2% agarose gel using a 100bp ladder for visualization

All primer combinations tested worked with bands visible in each lane! The strange band pattern seen in lane 6 is most probably due to non-specific binding.

Experimental Design – Primer combinations:

Template DNA	Fwd Primer	Rvs primer
DNA2.2	(AT-FWD-1)	(AT-RVS-1)
PCR product (from DNA2.2)	(AT-FWD-1)	(AT-RVS-1)
DNA2.2	(AT-BHO-F)	(AT-RVS-1)
PCR product (from DNA2.2)	(AT-BHO-F)	(AT-RVS-1)
DNA2.2	(AT-FWD-RBS)	(AT-RVS-1)
PCR product (from DNA2.2)	(AT-FWD-RBS)	(AT-RVS-1)
DNA2.2	(AT-FWD-1)	(AT-AHO-R)
PCR product (from DNA2.2)	(AT-FWD-1)	(AT-AHO-R)
DNA2.2	(AT-BHO-F)	(AT-AHO-R)
PCR product (from DNA2.2)	(AT-BHO-F)	(AT-AHO-R)
DNA2.2	(AT-FWD-RBS)	(AT-AHO-R)
PCR product (from DNA2.2)	(AT-FWD-RBS)	(AT-AHO-R)
ssH2O (-) control	(AT-FWD-1)	(AT-RVS-1)

Figure 3. PCR optimization results

Figure 3. GelRed post-stained 2% agarose gel of optimized PCR of A. tumefaciens bacteriophytochrome using various primers. In lanes 1 and 15 there is a 100bp ladder. In lane 2 is the DNA2.2 sample with (AT-FWD-1) and (AT-RVS-1)primer pair. In lane 3 there is PCR product template (from DNA2.2 sample) with (AT-FWD-1)and (AT-RVS-1)primer pair. In lane 4 there is DNA sample with (AT-BHO-F) and (AT-RVS-1)primer pair. In lane 5 is the PCR product (from DNA2.2 sample) with (AT-BHO-F)and (AT-RVS-1)primer pair. In lane 6 there is DNA2.2 sample with (AT-FWD-RBS)and (AT-RVS-1)primer pair. In lane 7 there is PCR product template (from DNA2.2 sample) with (AT-FWD-RBS)and (AT-RVS-1)primer pair. In lane 8 there is DNA2.2 sample with (AT-FWD-1) and (AT-AHO-R)primer pairs. In lane 9 there is PCR product template (from DNA2.2 sample) with (AT-FWD-1) and (AT-AHO-R)primer pairs. In lane 10 there is DNA2.2 sample with (AT-BHO-F) and (AT-AHO-R)primer pairs. In lane 11 there is PCR product (from DNA2.2 sample) with (AT-BHO-F)and (AT-AHO-R)primer pair. In lane 12 there is DNA2.2 sample with (AT-BHO-F)and (AT-AHO-R)primer pair. In lane 13 there is PCR product (from DNA2.2 sample) with (AT-FWD-RBS)and (AT-AHO-R)primer pair. In lane 14 there is ssH2O negative control with (AT-FWD-1)and (AT-RVS-1)primer pair. All the lanes worked. There is a weird result in lane 7 which is probably due to non-specific binding. = SUCCESS!

3rd September 2010
PCR Optimization (using Gradient PCR)

Now that we have the A. tumefaciens bacteriophytochrome product as well as the RBS inserted it is now time for us to amplify the bacteriophytochrome product with the heme oxygenase gene

We will be inserting the two genes in two different orientations in the operon

We will achieve this using two different primer pairs

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 65C

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.

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.

A day-by-day progress for Deinococcus Radiodurans Bacteriophytochrome Operon Construct

16th September 2010

Genomic DNA extraction

D. radiodurans genomic DNA extracted using the BioLine Genomic DNA Extraction kit as per the manufacturer’s protocols.

The extractions were run on a GelRed stained 1% agarose gel and photo taken for visualization (see figure 6).

A NanoDrop spectrophotometer reading was also recorded to check the quality of the extracted genomic DNA.

The extraction was successful for all D. radiodurans cell lysate samples (labeled DEINO1, DEINO2, DEINO3 (See figure below)

D. radiodurans genomic DNA extraction agarose results:

All three DNA samples show a smear of gDNA. Because the samples were treated with RNase there is no band indicative of RNA visible = SUCCESS!

Figure 6. Results of D. radiodurans genomic DNA extraction

***********FIGURE6************

Figure 6. GelRed post-stained 1% agarose gel of genomic DNA extraction from D. radiodurans . In lanes 1 and 5 there is a 1kb ladder. In lane 2 is the DEINO1 sample, lane 3 is the DEINO2 sample, lane 4 is the DEINO3 sample. All three samples show a smear that is indicative of genomic DNA. The extraction has been successful!!!

Nanodrop absorbance readings:

Genomic DNA sample	260/280 OD ratio	Concentration (ng/mL)
DEINO1	2.43	1.443
DEINO2	2.55	51.8
DEINO3	2.99	88.7

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

= SUCCESS!

27th August 2010
Primer design

Various primers were designed manually and using Primer 3 Software package for PCR amplification.

The primers were ordered and supplied through Integrated DNA Technologies.

There was an array of various primers ordered for amplification of different products. The details of the primers are described below.

Fwd and Rvs primers for amplification of the full length D. radiodurans BphP gene:

Primer name	Primer Sequence
(DR-FWD-1)	5’-ATG AGC CGG GAC CCG TTG -3’
(DR-RVS-1)	5’-TCA GGC ATC GGC GGC TCC -3’

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

Primer name	Primer Sequence
(DR-BHO-F)	5’- AAG GAG ATA TAC ATA TGA TGA GCC GGG ACC CGT TG – 3’
(DR-BHO-R)	5’- AAG TTG ACA CTC ATA TGA GCA GCC CTC CTT CAG GC – 3’

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

Primer name	Primer Sequence
(DR-AHO-F)	5’- CCG AAG GCT AGG ATC CAG GAG GGC TGC TAT GAG C – 3’
(DR-AHO-R)	5’- GTT AGC AGC CGG ATC CTC AGG CAT GGG CGG CTC C – 3’

Fwd and Rvs primers for amplification of the full length D. radiodurans 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:

Primer name	Primer Sequence
(DR-FWD-RBS)	5’- AGG AGG GCT GCT ATG AGC CGG GAC CCG TTG -3’

22nd September 2010
Initial PCR (gradient PCR)

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
55-65˚C for 30 seconds
72˚C for 2 minutes & 30 seconds

(This was repeated for another 25 cycles)

72˚C for 5 minutes
4˚C to end.

Different combinations of the primers were used for the initial PCR reaction (see below ‘Experimental Design’ section following)

Not all possible primer combinations were used

The PCR products were run on a 2% GelRed post-stained agarose gel for visualisation (see picture of gel below).

Experimental Design – Primer combinations and annealing temperatures:

DNA template	Dilution	Fwd primer	Rvs primer	Annealing temp (Degrees Celsius)
DEINO1	1:20	(DR-FWD)	(DR-RVS)	55, 57, 60, 62, 65
DEINO2	1:20	(DR-FWD)	(DR-RVS)	55, 57, 60, 62, 65
DEINO1	1:20	(DR-FWD-RBS)	(DR-RVS)	55, 57, 60, 62, 65
DEINO2	1:20	(DR-FWD-RBS)	(DR-RVS)	55, 57, 60, 62, 65
DEINO1	1:20	(DR-FWD)	(DR-AHO-R)	55, 57, 60, 62, 65
DEINO2	1:20	(DR-FWD)	(DR-AHO-R)	55, 57, 60, 62, 65
DEINO1	1:20	(DR-FWD-RBS)	(DR-AHO-R)	55, 57, 60, 62, 65
DEINO2	1:20	(DR-FWD-RBS)	(DR-AHO-R)	55, 57, 60, 62, 65
DEINO1	1:50	(DR-FWD)	(DR-RVS)	55, 57, 60, 62, 65
DEINO2	1:50	(DR-FWD)	(DR-RVS)	55, 57, 60, 62, 65
DEINO1	1:50	(DR-FWD-RBS)	(DR-RVS)	55, 57, 60, 62, 65
DEINO2	1:50	(DR-FWD-RBS)	(DR-RVS)	55, 57, 60, 62, 65
DEINO1	1:50	(DR-FWD)	(DR-AHO-R)	55, 57, 60, 62, 65
DEINO2	1:50	(DR-FWD)	(DR-AHO-R)	55, 57, 60, 62, 65
DEINO1	1:50	(DR-FWD-RBS)	(DR-AHO-R)	55, 57, 60, 62, 65
DEINO2	1:50	(DR-FWD-RBS)	(DR-AHO-R)	55, 57, 60, 62, 65
(-) control	-	(DR-FWD)	(DR-RVS)	55, 57, 60, 62, 65

All 85 reactions were unsuccessful and no bands were visible on the gel = BACK TO THE DRAWING BOARD!

27th September 2010
PCR with FailSafeTM PCR system

As the 85 reactions that were run were unsuccessful we decided to purchase a commercial PCR system called the FailSafeTM System from EpiCentre Biotechnologies

It was speculated that the amplification didn’t work due to either the enzyme / buffer or the combination. It didn’t seem to be the annealing temperatures because a range of temperatures were tested in the previous PCR

The FailSafeTM PCR System includes 12 different enzyme / buffer combinations so we decided to try this with two different annealing temperatures (56˚C and 60˚C)

The PCR program was set up as per the following:

94˚C for 2 minutes
94˚C for 30 seconds
56˚C or 60˚C for 30 seconds
72˚C for 3 minutes

(This was repeated for another 25 cycles)

72˚C for 10 minutes
4˚C to end.

The PCR products were run on a GelRed stained 2% agarose gel using a 1kb ladder for visualization (see figure 2 below)

We have successfully amplified the D. radiodurans bacteriophytochrome gene! SUCCESS!

Experimental Design – Primer combinations:

Template DNA	Dilution	Fwd Primer	Rvs primer	FailSafe system
DEINO1	1:100	DR-FWD-1	DR-RVS-1	(All 12 FailSafe premixed combinations)
DEINO2	1:100	DR-FWD-1	DR-RVS-1	(All 12 FailSafe premixed combinations)

FailSafe PCRTM System results:

*******************FIGURE 7 ***********

Figure 7. GelRed post-stained 2% agarose gel of FailSafeTM PCR System. In lanes 1, 15, 16 and 30 there is a 1kb ladder. In lanes 2 to 29 is the D. radiodurans DNA template amplified with the (DR-FWD-1) and (DR-RVS-1) primer pair using the 12 different premixed enzyme / buffer combinations from the FailSafeTM PCR System. The top lanes (2-15) have a 56˚C annealing temperature. The bottom rows have a 60˚C annealing temperature. It is obvious by looking at the gel that there are differences in the non-specific binding patterns using the different temperature, enzyme and buffer combinations.

8th October 2010
PCR (with FailSafeTM PCR System and DR-FWD-RBS primer)

We used the FailSafeTM PCR System to amplify the D. radiodurans bacteriophytochrome with the additional RBS inserted (using DR-FWD-RBS primer)

The most successful enzyme/buffer combinations (J & K) from the previous PCR were used with an annealing temperature of 60C for this amplification

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 3 minutes

(This was repeated for another 25 cycles)

72˚C for 10 minutes
4˚C to end.

The PCR products were run on a GelRed stained 2% agarose gel using a 1kb ladder for visualization (see figure 3 below)

We have successfully amplified the D. radiodurans bacteriophytochrome gene with the additional RBS site! SUCCESS!

PCR results using DR-FWD-RBS primer with FailSafe PCR system:

*******************FIGURE 8 ***********

Figure 8. GelRed post-stained 2% agarose gel of FailSafeTM PCR System. In lanes 1 and 5 there is a 1kb ladder. In lane 2 there is the XXX PCR product amplified with the (DR-FWD-RBS) and (DR-RVS-1) primer pair using buffer J from the FailSafeTM PCR System. In lane 3 there is the XXX PCR product amplified with the (DR-FWD-RBS) and (DR-RVS-1) primer pair using buffer K from the FailSafeTM PCR System.

9th October 2010
PCR with FailSafeTM PCR System and (DR-AHO-F) and (DR-AHO-R) primer pair

We used the FailSafeTM PCR System to amplify the D. radiodurans bacteriophytochrome with the additional RBS product obtained from the previous PCR using the (DR-AHO-F) and (DR-AHO-R) primer pair to insert the HO site

Again, the most successful enzyme/buffer combinations (J & K) from the previous PCR were used with an annealing temperature of 60˚C for this amplification

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 3 minutes

(This was repeated for another 35 cycles)

72˚C for 10 minutes
4˚C to end.

The PCR products were run on a GelRed stained 2% agarose gel using a 1kb ladder for visualization (see figure 3 below)

We have successfully amplified the D. radiodurans bacteriophytochrome gene with the additional RBS site as well as the HO site! SUCCESS!

We are ready for cloning!

PCR results of bacteriophytochrome and RBS template using (DR-AHO-F) and (DR-AHO-R) primer pair with FailSafeTM PCR System:

*******************FIGURE 9 ***********

Figure 9. GelRed post-stained 2% agarose gel of FailSafeTM PCR System. In lanes 1 and 5 there is a 1kb ladder. In lane 2 there is the 1st PCR product (bacteriophytochrome and RBS) amplified with the (DR-AHO-F) and (DR-AHO-R) primer pair using buffer J from the FailSafeTM PCR System. In lane 3 there is the 2nd PCR product (bacteriophytochrome and RBS) amplified with the (DR-AHO-F) and (DR-AHO-R) primer pair using buffer K from the FailSafeTM PCR System. We have successfully amplified the D. radiodurans bacteriophytochrome gene with both the RBS and HO site inserted. This is now ready for cloning!!

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 <center><h2> A day-by-day progress for <i> Agrobacterium Tumefaciens </i> Bacteriophytochrome </h2>   </center>
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 Overall, the Nanodrop readings show that we have obtained good DNA concentration with minimal protein contamination <p>= <u>SUCCESS! </u><p>
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 <li type="disc">The reaction mastermix for the initial PCR was set up as per the following recipe (per sample): </li>
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-PCR Optimization<p> </big> </hr> </font> </div>
+PCR Optimization<p> </big> </hr> </font> </b>
 <li type="disc"> Now that we have a product obtained by the initial PCR it is time to optimize the PCR conditions using the successful DNA2.2 PCR product obtained from last week and the original DNA2.2 template </li>
 <li type="disc"> The reaction mastermix for the PCR was set up as per the following recipe (per sample): </li>
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 rd September 2010  <p>
-PCR Optimization (using Gradient PCR)<p> </big> </hr> <p> </font></div>
+PCR Optimization (using Gradient PCR)<p> </big> </hr> <p> </font></b>
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 <li type="disc">The reaction mastermix for the PCR was set up as per the following recipe (per sample): </li>
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-Genomic DNA extraction <p> </big> </font> </div>
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 <menu>
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 <h4>Nanodrop absorbance readings: </h4>
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 </table>
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-<p> </div>
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 Overall, the Nanodrop readings show that we have obtained good DNA concentration with minimal protein contamination <p>= <u>SUCCESS! </u><p>
 <p><p>
-<big><hr>
+<big><hr><b>
 th August 2010 <p>
-Primer design <p> </big> </hr>
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 <h4>Fwd and Rvs primers for amplification of the full length <i> D. radiodurans </i> BphP gene: </h4>
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-Initial PCR (gradient PCR)<p> </big> </hr> </font> </div>
+Initial PCR (gradient PCR)<p> </big> </hr> </font> </b>
 <li type="disc"> The reaction mastermix for the PCR was set up as per the following recipe (per sample): </li>
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 th September 2010 <p>
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 <p><h3><b> FailSafe PCRTM System results: </p></B></h3>
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-PCR (with FailSafeTM PCR System and DR-FWD-RBS primer)<p> </big> </hr> <p> </font></div>
+PCR (with FailSafeTM PCR System and DR-FWD-RBS primer)<p> </big> </hr> <p> </font></b>
 <li type="disc">We used the FailSafeTM PCR System to amplify the D. radiodurans bacteriophytochrome with the additional RBS inserted (using DR-FWD-RBS primer)</li>
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-PCR with FailSafeTM PCR System and (DR-AHO-F) and (DR-AHO-R) primer pair<p> </big> </hr> <p> </font></div>
+PCR with FailSafeTM PCR System and (DR-AHO-F) and (DR-AHO-R) primer pair<p> </big> </hr> <p> </font></b>

Team:Macquarie Australia/Notebook

From 2010.igem.org

Revision as of 21:31, 19 October 2010

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

A. tumefaciens genomic DNA extraction agarose results:

Figure 1. Results of A. tumefaciens genomic DNA extraction

Figure 1. Results of A. tumefaciens genomic DNA extraction

Nanodrop absorbance readings:

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:

Experimental Design – Primer combinations:

Figure 2. Results of the initial PCR

The PCR program was set up as per the following:

Experimental Design – Primer combinations:

Figure 3. PCR optimization results

The PCR program was set up as per the following:

Experimental Design – Primer combinations and annealing temperatures:

Figure 4. PCR Optimisation (gradient PCR) results

The PCR program was set up as per the following:

Experimental Design – Primer combinations and annealing temperatures:

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

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):

Experimental Design – Primer combinations and annealing temperatures:

Figure 5. PCR optimization (with ssPCR amplification for (AT-BHO-F)primer)

A day-by-day progress for Deinococcus Radiodurans Bacteriophytochrome Operon Construct

D. radiodurans genomic DNA extraction agarose results:

Figure 6. Results of D. radiodurans genomic DNA extraction

Figure 6. Results of D. radiodurans genomic DNA extraction

Nanodrop absorbance readings:

Fwd and Rvs primers for amplification of the full length D. radiodurans BphP gene:

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

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

Fwd and Rvs primers for amplification of the full length D. radiodurans 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:

The PCR program was set up as per the following:

Experimental Design – Primer combinations and annealing temperatures:

The PCR program was set up as per the following:

Experimental Design – Primer combinations:

FailSafe PCRTM System results:

The PCR program was set up as per the following:

PCR results using DR-FWD-RBS primer with FailSafe PCR system:

The PCR program was set up as per the following:

PCR results of bacteriophytochrome and RBS template using (DR-AHO-F) and (DR-AHO-R) primer pair with FailSafeTM PCR System:

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.