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:

*DNA*
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.

TEAM COMPETENT CELLS

-Competent Cell Method Modified from Inoue et al. 1992- Colonies isolated and inoculated to 150ml of SOB medium and grown to an A600 of between 0.2-0.8 at room temperature overnight (18-22 C) with vigorous shaking (200-250 rpm), then place on ice for 10min. The culture was transferred to a 15 ml centrifuge tube and spun at 2500 x g for 10 min at 4 C. Cells pelleted by spinning the tubes at ~2,500 g for ~7 min at 4oC in the Sigma centrifuge. Supernatant removed, and immediately the tube was placed on ice. Cells were resuspended in 1-5 ml of ice-cold TB buffer by gently pipetting up and down with a 1 ml pipette. Ice-cold TB buffer was added to bring the volume up to ~1/5th of the original culture volume, and mixed by gently inverting. Tube was incubated on ice for 10 min. Centrifugation step repeated, supernatant poured off and cells gently resuspend in ~1/20th of the original culture volume of ice-cold TB buffer. 930 l of your cell suspension added to 1.5 microcentrifuges. 70 l of DMSO added to each. Aliquots of 100 *l of the competent cell suspension were combined with DMSO into microcentrifuge tubes. Competent cell aliquots where then frozen in liquid nitrogen. In order to prepare and transform competent E.coli cells various nutrient broths, such as LB, LB agar, SOC and SOB medium, were prepared.

MEDIA PREP

-LB media Method- Dissolve 10g tryptone, 5g yeast extract and 10g NaCl in 800 mL MilliQ water, making use of a magnetic stirrer. Once dissolved, bring volume up to 1 liter 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 refrigerator.

SOC and SOB medium are known to result in higher transformation efficiencies of plasmids (1).

-SOC media (for competent cells)- A mixture of 4g 2% w/v bacto-tryptone (20 g), 1g 0.5% w/v bacto-yeast extract, 400µl 5M NaCl, 250 µl 2M KCl, 20mM MgSO4 (0.4821 g), 0.7222g 20mM glucose and 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.

-SOB Media (for competent cells)- Distilled H2O of volume 900ml was added with 5g Bacto Tryptone, 1.25g Bacto Yeast Extract, 0.124 grams NaCl and 0.0475 grams KCl. This was then adjust to pH 7.0 with NaOH or HCl and then to 250mL with distilled H2O. The system was then sterilized by autoclaving.

BUFFER PREP

TB buffer (for competent cells)- 2 grams of CaCl2-2H2O, 18.6 grams of KCl and 3 grams of PIPES where mixed and dissolved in ca 500ml of water with the pH adjusted to 6.7 with KOH. Then, 10.9 grams of MnCl2-4H2O, isdissolved in 300 ml of water, mixed and the solution adjusted to 1L. This is then sterilized by filtration through a pre-rinsed 0.45 µm filter unit and stored at 4 C. The solution is stable at 4 C indefinitely. All salts are added as solids. Resulting solution was colourless and unautoclaved.

19/8/2011

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

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

@@ Line 91: / Line 91: @@
 :::{| border="1" cellpadding="10" cellspacing="0"
-|+ align="bottom" style="color:#e76700;" |''Ch vector prep'
+|+ align="bottom" style="color:#e76700;" |''Ch vector prep''
 |-
 |'''Rectants'''
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 :::{| border="1" cellpadding="10" cellspacing="0"
-|+ align="bottom" style="color:#e76700;" |''Nanodrop'
+|+ align="bottom" style="color:#e76700;" |''Nanodrop''
 |-
 |'''PCR product'''