Team:Macquarie Australia/Notebook

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Notebook

12/8/2011

Team PCR

Today, we started the PCR process in order to amplify our T7 promoter, HO1, At, Dr.

• Step 1) We diluted the primer stock down to a concentration of 100uM. We did this by taking the weight of the primers and multiplying them by 100 and then adding this amount of water to the stock.

So, for example, if our amount of Oligo = 38.3nM, we add 383ul of water (which gives us a concentration of 100uM)

• Step 2) We performed a second dilution (1:10) to produce a concentration of 10uM for each primer.

So, 100uM --> 10uM

• Step 3) A master-mix was made containing:

Master Mix

Reactants	Volume
Water	41.4ul
5x buffer	16ul
10mM DNTP	1.6ul
Taq poly	1ul

• Step 4) Mix was spun for a couple of seconds. This master mix as divided up into 4 15ul aliquots [you can take out 3 lots of 15ul and keep the final 15ul in the mastermix tube and use that as a housing].

• Step 5) To these 15ul aliquots, we add into each:

Master Mix

Primers/Template (steps 1-2)	Volume
Forward	2ul
Reverse	2ul
Template [Ho, Dr etc]	1ul

What we end up with is 4 reaction tubes with our plasmid template with the primers in the correct dilution.

• Step 6) We set up the PCR machine with the following protocol:

Initial denaturation – 94oC for 2 minutes

Denaturation – 94oC for 30 sec

Annealing – 60oC for 30 seconds ] 10 cycles in total

Extension – 72oC for 2min 30sec

THEN

Denaturation – 94oC for 30 sec

Annealing – 52oC for 30 seconds ] 15 cycles in total.

Extension – 72oC for 2min 30sec

Final extension – 72oC for 10 min

Hold period – 4oC for 2 minutes

We’ve labelled the protocol ‘330’ and it’s the same as last years’ protocol with a few additions. Note that we made a mistake during our 15 cycle stage - it should be at 72oC. Our bad. We have fixed this - the protocol has been corrected.

• Step 7) We conducted our PCR! It took 2 hours and 13 minutes to complete.

• Step 8) With our PCR complete, we then set about preparing the Chloramphenicol vector [which is the one we need to submit for any medal] as well as the clean up protocol.

Ch vector prep'

Rectants	Volume
DPN1	1ul
Xba1 + Spe1	1ul + 1ul
100x BSA	1ul
Template DNA - Ch vector	20ul
10x NEBuffer 4	5ul
Water	21.5ul
Total	50.5ul

Clean up prep:

Sigma GENELUTE PCR clean up kit has the protocol

• Step 9) We set up the nanodrop to determine the quality of our PCR results:

Nanodrop'

PCR product	Concentration
T7	1.1ug [23ng/ul]
HO	870ng [17.4ng/ul]
Agro	500ng [10.4ng/ul]
Dino	1.8ug [36.1ng/ul]

• Step 10) Cleaned PCR products were diluted with water [20ul PCR product + 30ul water] and were then double digested using X + S [1ul each] in 5ul of 10x buffer and 1ul of BSA.

Media Prep

-LB media Method- Dissolve 10g trytone, 5g yeast extract and 10g NaCl in 800 mL MilliQ water, making use of a magnetic stirrer. Once dissolved, bring volume up to 1 litre using the MilliQ water. Autoclave 500 mL of the solution (121oC, 15 min, standard liquid cycle).

-LB agar Method- Add 7.5 g Bacto agar to the remaining 500 mL of LB media and autoclave [121oC, 15 min, standard liquid cycle]. Add 250 µL of Chloroamphenicol and mix well before plating out and setting agar.

After cooling and antibiotic addition, the LB agar was plated out using aseptic technique. 14 LB agar plates were prepared and allowed to cool next to the bunsen. After this all plates were aseptically sealed using parafilm and stored in the refridegerator.

SOC prep

In order to prepare and transform competent E.coli cells various nutrient broths, such as LB, LB agar, SOC and SOB medium, were prepared. SOC and SOB medium are known to result in higher transformation efficiencies of plasmids (1). The SOC media was prepared using the following guidelines:

2% w/v bacto-tryptone (20 g)

0.5% w/v bacto-yeast extract (5 g)

8.56mM NaCl (0.5 g)

2.5mM KCl (0.186 g)

20mM MgSO4 (2.408 g)

20mM glucose (3.603 g)

Distilled H2O to 1000 mL (2)

It was decided that over the course of the project only 200ml of SOC media would be needed. Therefore the above values were adjusted to the following quantities:

2% w/v bacto-tryptone (4 g)

0.5% w/v bacto-yeast extract (1 g)

400 µl 5M NaCl

250 µl 2M KCl

20mM MgSO4 (0.4821 g)

20mM glucose (0.7222 g)

MilliQ H2O to 200 mL

The adjusted quantities were combined in 1l measuring column with constant stirring and then placed in the autoclave for sterilization.

1. Hanahan, D. (1983) J Mol Biol 166, 557-580

2. Sambrook, J. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed ed., Plainview, NY