Team:Glasgow/Notebook/Protocols

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Protocols

This page shows our protocols for various lab procedures:

• Creating Chemically Competent Cells
• Transformation of Competent Cells
• DNA Clean-up
• PCR
• Restriction Digests
• Gel Extraction
• Annealing Top and Bottom Strand Oligos
• Oligos Purification
• Site Directed Mutagenic PCR – Quikchange Protocol
• In vitro φC31 Integrase Reaction
• Ligations
• Drying DNA for Sequencing
• DNA Mini-prep
• E.coli Bacterial Strains Used

Creating Chemically Competent Cells

1. Pipette 400μl of culture into 20ml of fresh broth
2. Incubate the cultures in shaking 37⁰C water bath for 90 min
3. 50 mM Calcium chloride and centrifuge tubes must be cooled on ice.
4. Pour culture into centrifuge tubes, return to ice.
5. Centrifuge in a cold rotor for 2 min at 7000rpm (6000 G)
6. Pour out the supernatant, being careful about the pellet
7. Add 10μl of calcium chloride, immediately return to ice and leave for 1 hour (or longer)
8. Repeat the centrifuge step, pour out supernatant
9. Store on ice, add 1ml of calcium chloride. Resuspend and return to ice.

Transformation of Competent Cells

1. Add 100μl of competent cells to cooled Eppendorfs
2. Add 1μl of DNA
3. Incubate on ice for 20 min
4. Heat shock at 37⁰C for 5 min
5. Place immediately on ice and leave for 5 min.
6. Add 200μl of L-Broth and incubate at 37⁰C for 90 min. Expression step.
7. Spread 100-200μl on dried L-agar plates with required antibiotics. On half plates use 40μl.
8. Incubate plates

DNA Clean-up

1. Transfer supernatant to a mini-column
2. Spin for 1 min on full and pour out flowthrough
3. Add 500μl of PB, spin for 1 min and discard flowthrough
4. Add 800μl of PE, spin for 1 min and discard flowthrough
5. Spin again and discard flowthrough.
6. Add 50μl of EB to the centre of the white disc and stand for 1 min.
7. Move column to a 1.5ml Eppendorf (leaving behind the collection tube) and spin for 1 min.
8. Keep supernatant. DNA is in the liquid.

PCR

50μl reaction volume –

• 10μl 5 x HF Buffer
• 5μl 5mM dNTPs
• 1μl 50mM MgCl2
• 1.5μl DMSO
• 5μl 5μM F primer
• 5μl 5μM R Primer
• 22μl ddH2O
• 1μl (1/100 dilution) template DNA
• 0.5μl polymerase

Programme Settings

1. 98⁰C – 1 min
2. 98⁰C – 20 sec
3. 55⁰C – 30 sec
4. 55⁰C – 30 sec (Back to step 2. x30)
5. 72⁰C – 10 min
6. 4⁰C – Hold

Restriction Digests

1. Add
   a. For a 20μl sample
   1. 2μl 10 x buffer
   2. 4μl DNA sample
   3. 14μl ddH20
   4. 0.5μl restriction enzyme
   b. For a 30μl sample
   1. 3μl 10 x buffer
   2. 10μl DNA sample
   3. 17μl ddH20
   4. 0.75μl restriction enzyme
2. Mix thoroughly
3. Incubate at 37⁰C for at least an hour

Gel Extraction

1. Place gel slice into a microfuge tube
2. Weigh microfuge tube, add 3 volumes of Buffer QG to volume of gel (100μg = ~100μl)
3. Incubate at 50⁰C for 10 min (until gel has dissolved) mix by vortex every 2-3 min in incubation
4. Add 1 gel volume of isopropanol to sample and mix.
5. Apply sample to spin column, centrifuge 1 min and discard flowthrough.
6. Add 500μl Buffer QG and centrifuge for 1 min.
7. Add 750μl Buffer PE, stand for 2-5 min and centrifuge for 1 min.
8. Discard flowthrough and centrifuge again for 1 min.
9. Move spin column to microfuge tube
10. Add 30μl Buffer EB to centre of white disc stand for 1 min and centrifuge for 1 min.
11. Keep supernatant.

Annealing Top and Bottom Strand Oligos

Mix-

• 10μl top strand 100μM
• 10μl bottom strand 100μM
• 80μl ddH2O

To Anneal-

1. Heat in 85⁰C metal heating block for 5 min
2. Then turn off heat block leaving tube in block to cool down slowly (1-2 hours) to ~30⁰C
3. Dilute ‘annealed’ (cold) oligos - 1μl oligo and 99μl ddH2O

Oligos Purification

1. Prepare the polyacrylamide gel -
   a. Prepare the following –
   1. Urea – 18.2g
   2. 40% acrylamide – 8ml
   3. Add ddH20 up to 40ml
   b. Mix completely
   c. Add 480μl of 10% Ammonium Peroxidisulphate
   d. Add 24μl of TEMED
This creates a 1mm thick gel.
2. Pre-run the gel at 400V and 30mmh to warm up
3. Prepare the samples by heating at 80⁰C on a heating block for 5 min and then add equal volume formamide loading buffer to sample volume.
4. Run the gel at 400v for 90 min
5. Stain the gel in the following for 5 min –
   a. 70ml ddH20
   b. 20ml isopropanol
   c. 10ml “stains all”
6. Cut gel. Cut out the strongest full size oligo-bands and put in a labelled Eppendorf tube
7. Crush gel fragments
8. Add 500μl of TE
9. Put in a shaker at 37⁰C at 1150 overnight
10. Spin down at 10, 000 rpm for 1 min
11. Transfer the supernatant to a 0.22 coster filter
12. Resuspend the pellet with 100μl TE
13. Spin down supernatant for 1 min at 10, 000 rpm
14. Spin filter for 1 min at 3, 000 rpm
15. Transfer to Eppendorf tube
16. Dry vacuum for ~3 hours at 20⁰C to 200μl
17. Add 1/9 of the sample volume of Na acetate to the tube
18. Add 2.5 sample volumes of 100% ethanol to the tube
19. Mix well and store at -20⁰C overnight
20. Spin at full speed at -20⁰C for 30 min
21. Add 1ml of 80% ethanol after removing the supernatant without disturbing the pellet
22. Spin for 5 min at full speed
23. Remove the supernatant. If the pellet is disturbed, keep the removed supernatant in a separate Eppendorf.
24. Spin for 30 seconds at full speed then remove the final bit of supernatant.
25. Leave open at room temperature to dry for 5-10 min.
26. Add 20μl of 0.1 x TE and leave to dissolve for ~30 min.
27. Calculate the oligo concentration through the use of a spectrophotometer.

Site Directed Mutagenic PCR – Quikchange Protocol

1. Prepare the 25μl reaction as below –
   1. 2.5μl of 10 x Pfu buffer
   2. 1μl (of a 1/10 dilution) plasmid template
   3. ~62.5ng or 6pmol top primer (1/10 dilution)
   4. ~62.5ng or 6pmol bottom primer (1/10 dilution)
   5. 2.5μl 2mM dNTP mix
   6. 15-17μl ddH20
2. Add 0.5μl of Pfu-turbo polymerase
3. Run with the following PCR programme –
   1. 95⁰C for 1 min
   2. 95⁰C for 40 sec
   3. 53⁰C for 40 sec
   4. 68⁰C for 14 min
   5. Go back to step b and repeat 15 times
   6. 4⁰C hold
4. Set up the following restriction digest –
   1. 10μl of Quikchange reaction
   2. 1μl of Buffer A
   3. 9μl of ddH20
   4. 1μl of DpnI
5. Vortex and then incubate at 37⁰C for 2 hours
6. Transform 1μl of reaction

In vitro φC31 Integrase Reaction

20µl sample

1. Add
   • 5µl 4 x IRB5 buffer
   • 4 µl plasmid DNA
   • 2 µl integrase
   • 11 µl ddH2O
   or
   • 14 µl 1.43 x IRB3
   • 4 µl plasmid DNA
   • 2 µl integrase
2. Incubate at 30°C for 1hr 15min – 2hrs
3. Heat at 78 – 80 °C for 10mins (to kill enzyme)
4. Spin down

Ligations

10 µl sample

1. Add:
   • 1 µl 10x ligation buffer
   • 4 µl Insert
   • 5 µl Vector
   • 0.5 µl ligase
   or
   • 5μl of gel purified vector
   • 1μl (of 1/100 dilution) of annealed oligos (or ddH2O control) to make a 10mM oligo solution
   • 1μl of 10 x ligation buffer
   • 3μl of ddH20
   • 0.5μl ligase
2. Vortex
3. Leave at room temperature overnight

Drying DNA for Sequencing

1. Add 8 µl of DNA sample to 1.5 µl Eppendorf
2. Dry DNA in Vacuum Dryer Centrifuge for 30min to 1 hour (with lid open)
3. In separate tubes, add 1.5 µl of primer for 1st sequencing sample and 5 µl of primer for each reaction after (5 µl x n + 10 µl) n=No. of sequencing reactions

Concentration needed for primers
VF	10pmol/µl
VR	10pmol/µl

DNA Mini-prep

1. Add 1.4ml of cell culture to 1.5ml Eppendorf, spin at full speed for 2min.
2. Pour out supernatant. Repeat this with another 1.4ml of culture.
3. Remove any remaining broth.
4. Add 250 µl of P1 and resuspend.
5. Add 250 µl of P2, invert 6 times to mix and leave for 2 min.
6. Add 350 µl of N3 and invert 6-8 times.
7. Spin on full for 10 min.
8. Keep supernatant. DNA is in the liquid.
9. Carry out DNA Clean-up as above using Qiagen miniprep columns.

E.coli Bacterial Strains Used

• DS941 is AB1157 recF, lacIq lacZ Delta M15
• DS941-Z1 as DS941 but with tet repressor and extra copies of lacI
• Top10
• DH5 α
• DH5 α

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