Molecular biology protocols

(in C. Dauphin-Villemant lab, 2008)

PCR
(using Go Taq from Promega)

in 25mL:
5uL 5x buffer (does not contain MgCl2)
2uL MgCl2 25mM
14.375uL H2O
1uL   genomic DNA
1uL dNTP mix (2.5mM)
1.5uL primer mix at a 10uM dilution each (keep also a 100uM dilution stock at –20∞C: if 9.19 nmoles of lyophilized oligonucleotides: add 92uL of H2O milliQ)
0.125uL GoTaq polymerase

        2min 94∞C
       30s 94∞C
35 times:   30s 48-60∞C depending on primers
       x min   72∞C (1kb/min)
       8 min 72∞C (to terminate all the sequences)
       4∞C for ever
check 5uL on gel

Gel extraction of PCR products
(using Promega Wizard SV Gel and PCR Clean-Up System)
1. excise DNA band from gel and place in a 1.5mL microcentrifuge tube.
2. weigh the gel (usually around 100-200mg). Add 1uL of Membrane Binding Solution for 1mg of gel. Incubate for about 5-10min at 50-65C until the gel is completely disolved.
3. transfer gel mixture to the minicolumn assembly. Incubate at RT for 1min.
4. centrifuge at 16,000g for 1min. Discard flowthrough and reinsert Minicolumn into Collection Tube.
5. add 700uL of Membrane Wash Solution (ethanol added). Centrifuge at 16,000g for 1min.
6. discard flowthrough and reinsert Minicolumn into Collection Tube.
7. add 500uL of Membrane Wash Solution (ethanol added). Centrifuge at 16,000g for 1min.
8. discard flowthrough and reinsert Minicolumn into Collection Tube.
9. recentrifuge for 5min at 16,000g with the microcentrifuge lid open (or leave your columns under the hood for 30min), so that ethanol is completely evaporated.
10. carefully transfer Minicolumn to a clean 1.5mL tube, add 50uL of nuclease-free water to the minicolumn. Incubate at RT for 1min.
11. centrifuge at 16,000g for 1min. Discard Minicolumn.
12. Let the ethanol evaporates fully by putting the tubes in the speedvac for 5min.
13. check 5uL on gel. The liquid should not go out of the wells if there is no ethanol left.

Preparation of Petri dishes with LB+ampicillin

1. prepare 500mL of LB with agar: (you can do about 4-5 plates with 100mL)
10g tryptone
10g NaCl
5g yeast extract
15g bacto-agar (bottle with “for bacteria” label)

stir well for about 30min
2. autoclave.
3. wait until the temperature is cold enough so that it would be possible to hold the bottle strongly with arms. Add ampicillin at a 1/1000 dilution (tubes stored at –20C in the brown cardboard box, top drawer, freezer C3).
4. pour in Petri dishes and wait about 10-20min so that it solidifies.
5. put the Petri dishes under the hood for about 30min-1h with the lid slightly open to dry them up.
6. store at 4C for about a month.

cloning of PCR products in pGEM-T Easy
before you start:
- put Petri dishes with LB+ampicillin at room temperature. On each plate, spread a mix of 100uL IPTG (100mM stock at 4C) and 20uL of X-Gal (50mg/mL in DMF, stock at -20C). Dry plates under the hood.
- have a 42C water bath ready
- make sure we have JM109 competent cells at -80C

1. set up the 5uL cloning reaction in a 1.5mL or 2mL tube:
   1.5uL of fresh or purified PCR product (if the PCR fragments have been resuspended in EB buffer, use 0.5uL PCR solution + 1.5uL water because the cloning reaction does not work well with 2uL of EB buffer)
   2.5uL ligation buffer 2x (vortex before use) (the box is at –80C)
   0.5uL pGEM-T Easy vector (50ng)
   0.5mL T4 DNA ligase
2. mix very gently with the pipet tip and incubate for 1h at RT or ON at 4C.
3. defrost tubes of competent cells JM109 (stored at -80∞C in a green plastic box – one tube=200uL) on ice.
4. add 40uL of competent bacteria to each cloning reaction. Do not mix up and down.
5. incubate for 20min on ice.
6. put the Petri plates at RT.
7. heat-shock the cloning reaction+bacteria at 42∞C (water bath) for exactly 45s and put back on ice for about 2min.
8. add 250uL of SOC (under the hood -SOC tubes are stored at RT) and close the tube tightly.
9. shake tubes horizontally at 37∞C for 1h30 at 150rpm.
10. Put about 200mL on a plate.
11. Incubate ON at 37∞C.

Day 2: inoculation of a LB culture
12. put plates at 4C for 30min-1h if the X-gal blue color is pale. The big white colonies are likely to contain your insert of interest.
13. prepare LB+ampicillin (dilute ampicillin stock (stored at -20∞C) at 1/1000)
14. prepare 1.5uL tubes with about 200L of LB+ ampicillin (under the hood)
15. prepare PCR mix: for each tube:
       5uL 5x buffer (does not contain MgCl2)
       2uL MgCl2 25mM
       14.725uL H2O
       1uL dNTP mix (2.5mM)
       1uL T7 primer (10pmoles/uL = 10uM dilution, at -20C in the primer box, third drawer)
       1uL Sp6 primer (10pmoles/uL = 10uM dilution, at -20C in the primer box, third drawer)
       0.125uL GoTaq polymerase
and store tubes on ice.
16. touch a white colonie with the pipet tip and then touch (1) the LB (under hood), (2) the PCR reaction tube and (3) a summary plate.
17. check 5uL of the PCR products on gel. The PCR product should be about 200bp longer than your insert.
18. prepare 15mL tubes with about 4mL of LB+ ampicillin (under the hood). Put 50uL of the LB+bacteria into each tube. Close falcon tube but leave space for air. Cover with tape, so that the cap doesn't go off.
19. incubate at 37∞C on agitation, not more than 14h.

Day Three: Mini-Prep (Wizard Plus SV Minipreps DNA Purification System)
20. centrifuge the 15mL tubes for 15min at 4500u/min. Remove supernatant.
21. thoroughly resuspend pellet with 250uL Cell Resuspension Solution and put the mixture into a 1.5mL tube.
22. add 250uL Cell Lysis Solution to each sample; invert 4 times to mix.
23. add 350uL Neutralization Solution; invert 4 times to mix.
24. centrifuge at full speed for 10min at RT. A compact white pellet will form.
25. put 750uL of the supernatant in a new tube. Centrifuge again at full speed for 10min at RT.
26. put 740uL of the supernatant in a new tube.
27. add the same volume of isopropanol (small brown glass bottle under the hood next to the -80C). Vortex and wait 2min at RT.
28. centrifuge at 15,000g at 4C for 5min.
29. Remove supernatant by inverting the tube and eliminate any drop of liquid by puting the tubes in the speedvac for 10min.
30. Wash pellet with 1mL of 70% ethanol stored at 4C.
31. Remove supernatant and dry the tube completely in the speedvac for at least 10min.
32. resuspend pellet in 100uL of nuclease-free water (Miniprep kit).
33. digest with EcoRI:
      16.5uL water
      2uL EcoRI buffer
      0.5uL EcoRI
      1uL Minniprep DNA
Incubate at 37C for about 1-2h. Put 5uL on a gel. Put also the low mass ladder (4uL+1uL water) on gel to estimate DNA concentration.
The Miniprep DNA should be around 100-200ng/uL.

Send DNA to Cogenics-Genome express for sequencing:
for each tube: 30uL of solution, with about 4ug DNA.

Glycerol stock:
pipet 340mL of the LB culture and add 60mL of sterile glycerol. Mix and store at -80∞C.

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Preparation of DIG-labeled probes

A) digestion of the plasmid
1. set up a digestion of at least 2ug of DNA:
10uL plasmid from the miniprep
4uL enzyme (SacI (+T7) or ApaI (+SP6))
5uL buffer (A for ApaI and J for SacI)
31uL water
   (50uL total)
incubate at 37C for 2-3h in the PCR machine.

B) purification of the linearized DNA
using the QIAquick gel extraction kit:
make sure everything is Rnase-free
1. add 25uL of RNase-free water to each sample
2. add 150uL of buffer QG
3. add 50uL of isopropanol, mix and apply the sample to a QIAquick column
4. centrifuge at 13 000rpm for 1min
5. discard flow-through
6. add 750uL of buffer PE, centrifuge at 13 000rpm for 1min
7. discard flow-through, centrifuge 1min
8. transfer the column to a new 1.5mL tube
9. add 30uL of buffer EB, wait 1min
10. centrifuge at 13 000rpm for 1min
11. check 1.5uL on gel to make sure that the digest is complete
(measure the DNA concentration if possible)

C) synthesis of labeled RNA
1. heat template DNA to 55C for 2min and put back on ice
2. set up the trancription reaction (in 20uL total):
9uL of template DNA (usually 1ug)
4uL Sp6/T7 5x buffer (Promega, stored at –20C in C4, top drawer, cardboard box)
2uL dNTP DIG RNA labeling mix (Roche, stored at –20C in C4, top drawer, Roche box)
2uL 100mM DTT (stored at –20C in C4, top drawer, cardboard box)
1uL RNAsine
2uL Sp6/T7 enzyme (stored at –20C in C4, top drawer, Roche box)
       (20uL total)

3. mix thoroughly and incubate at 37C for 2-2.5h in the PCR machine.
4. add 30uL RNAse-free DEPC water.
5. check 2uL on gel. The RNA band should be at least 10-fold stronger than the DNA template band.
6. store the reaction at –20C or proceed to the fragmentation step

D) fragmentation and precipitation
1. add 50uL of the carbonate buffer solution (stored at 4° C near the PCR machines)
2. mix and incubate at 65C for 20min (vary from 20 to 40min to control average probe fragment size)
3. add 100uL of the stop solution(stored at 4° C near the PCR machines = 0.3M NaOAc, 10% acetic acid, 10mM DTT)
4. put on ice right away.
5. add 20uL of acetate stop solution (acetate Na 3M pH=5, Falcon 15mL stored at 4° C)
+2uL DNA salmon sperm (blue plastic box, -20C, C3, drawer 1)
+500uL EtOH 100% RNase-free
6. vortex and freeze at –20C. Wait for at least 30min
7. spin at maximum speed for 20min at 4C.
8. wash pellet with 300uL 70% ethanol RNase free
9. spin at maximum speed for 2 minutes, drain completely and air dry using Speedvac
10. dissolve pellet in 200 µl hybridization solution or in 50uL DEPC water. Don't let the probe pellet dry for too long, it might become hard to resuspend. Let the probe dissolve on ice for a while, then mix it thoroughly by pipetting with a p200 and vortexing. Probe stocks should be stored at -20° C.