Team:Macquarie Australia/Notebook2

D. radiodurans genomic DNA extraction agarose results:

Figure 6. Results of D. radiodurans genomic DNA extraction

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:

1. 94˚C for 2 minutes
2. 94˚C for 30 seconds
3. 55-65˚C for 30 seconds
4. 72˚C for 2 minutes & 30 seconds

(This was repeated for another 25 cycles)

5. 72˚C for 5 minutes
6. 4˚C to end.

Experimental Design – Primer combinations and annealing temperatures:

The PCR program was set up as per the following:

1. 94˚C for 2 minutes
2. 94˚C for 30 seconds
3. 56˚C or 60˚C for 30 seconds
4. 72˚C for 3 minutes

(This was repeated for another 25 cycles)

5. 72˚C for 10 minutes
6. 4˚C to end.

Experimental Design – Primer combinations:

FailSafe PCRTM System results:

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.

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 3 minutes

(This was repeated for another 25 cycles)

5. 72˚C for 10 minutes
6. 4˚C to end.

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

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.

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 3 minutes

(This was repeated for another 35 cycles)

5. 72˚C for 10 minutes
6. 4˚C to end.

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

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!!