Molecular biology protocols

(used in A. Moore lab, 2006-2007)

Design of PCR primers
20 nucleotides long, 10 nmol scale (oligo kids) and standard purification
better in intronic than exonic region
melting temperature between 50 and 60∞C (http://www.basic.nwu.edu/biotools/oligocalc.html)
G/C at the 3’end
Use the temperature indicated on the invoice sheet (= roughly the one on the “oligonucleotide properties” website) for the PCR.

PCR from genomic DNA
(use Ex-Taq and Ex-buffer)

in 25mL:
2.5uL Ex buffer (contains 1.5 MgCl2, at –20C, top part, bottom right shelf)
16.25uL H2O (26.5uL if 10uL genomic DNA)
1uL   genomic DNA
2uL dNTP mix (at –20C, top part, bottom right shelf)
3uL 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)
0.25uL Taq polymerase

program #33, #34, #40:
       5min 94∞C

       30s 94∞C
30 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 2.5mL on gel

If necessary: gel extraction of the PCR fragments of correct size
(QIAquick PCR Purification Kit Protocol – Gel extraction kit QIAGEN #28706)
resuspend in 30uL of EB buffer

Preparation of Petri dishes with LB+kanamycine

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 kanamycine at a 1/1000 dilution (tubes at 20mg/mL stored at –20C in the orange box in the bottom left drawer, top part).
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.

TOPO TA cloning of PCR products
(Invitrogen #4506-40)

1. set up the 3mL cloning reaction in a 14mL Falcon tube:
        2mL 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)
0.5mL salt solution
       0.5mL TOPO vector (the box is at –20C top part in the bottom right drawer with restriction enzyme buffers)
2. mix very gently with the pipet tip and incubate for 5min at RT, then put tubes on ice.
3. defrost tubes of competent cells E. coli DH5a (stored at -80∞C in a light blue box, near the fly room – one tube=100uL) on ice.
4. add 30mL of competent bacteria to each cloning reaction and mix gently with pipet tip. Do not mix up and down.
5. incubate for 30min on ice.
6. put the Petri plates with kanamycine at RT.
7. heat-shock the cloning reaction+bacteria at 42∞C (water bath) for exactly 30s and put back on ice for about 5min.
8. add 240mL of SOC (flame, 1mL-SOC tubes are stored at –80C in the box with the competent cells) and close the tube tightly.
9. shake tubes horizontally at 37∞C for 1h at 250rpm.
10. Put about 200mL on a kanamycine plate.
11. Incubate ON at 37∞C.

Day 2: inoculation of a LB culture
12. prepare LB+kanamycine (dilute kanamycine stock (20mg/mL, stored at -20∞C in the orange box in the bottom left drawer, top part) at 1/1000)
13. add about 4mL of LB+ kanamycine into Falcon 14mL tubes (flame)
14. touch a white colonie with the pipet tip and then touch the LB (under hood). Close falcon tube but leave space for air.
15. incubate at 37∞C on agitation, not more than 14h.

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

QIAprep Spin Miniprep
(QIAprep Spin Miniprep QIAGEN #27106)

1. centrifugate 5min at 4∞C at 4,000rpm in 2mL tubes (load the tube twice to centrifugate 4mL).
2. remove supernatant.
3. resuspend the pellet with 250mL of Buffer P1 (stored at 4∞C in the fridge near the fly room) and transfer to a microcentrifuge tube. Ensure that RNAse A has been added to buffer P1. No cell clumps should be visible after suspension of the pellet.
4. add 250mL Buffer P2 and gently invert the tube 4-6times to mix. Do not vortex, do not allow the lysis reaction to proceed for more than 5 min.
5. add 350mL Buffer N3 and invert the tube immediately but gently 4-6 times. The solution should become cloudy.
6. centrifuge for 10min at 13,000rpm. A compact white pellet will form.
7. pour the supernatant into a QIAprep Spin Column. No white pellet   should go into the column, it does not matter if some liquid is discarded.
8. centrifuge for 1min. Discard the flow-through.
9. wash QIAprep Spin Column by adding 750mL Buffer PE and centrifuging for 1min.
10. discard the flow-through, and centrifuge for an additional 1min to remove residual buffer.
11. place the QIAprep column in a clean 1.5mL microcentrifuge tube. To elute DNA, add 50mL Buffer EB (10mM Tris-Hcl, pH 8.5) to the center of the QIAprep Spin Column, let stand for 1min, and centrifuge for 1min. The final DNA concentration is usually around 200-300ng/uL.
12. digest 5mL Miniprep DNA with EcoRI in 20mL total reaction: 2.5mL DNA
15mL H2O
2mL EcoRI buffer
0.5mL EcoRI
1h at 37∞C.
13. check 10mL of the reaction on a gel.

Sequence the fragment inserted into the TOPO vector
Prepare the mix reaction for sequencing in a 8-strip PCR tube:
0.5uL plasmid
1uL primer at 1.6pm/ul dilution (the M13 reverse or forward primers solution is in a green box at –20C, bottom left drawer with restriction enzymes)
3uL 5x dilution buffer (tube in yellow bag at 4C in the fridge near the fly room)
2uL BD terminator mix (at –20C in the box with M13 primers)
13.5uL water
(total = 20uL)

use the “msq” prgramm #1 on the PCR machine:
25 times:   10s    94∞C
       5s   50∞C
       4 min    60∞C
       4∞C for ever
wrap in aluminium foil and write name on it. Deposit in the fridge room S-008
on B1F of the BSI Central Building with the application sheet (check version1.1 on the application sheet).
http://s-008sv.brain.riken.jp/rrc-ga/dnaseq/index.htm
send also the Excel file by e-mail to rrc-ga@brain.riken.go.jp.

Preparation of DIG-labeled probes

A) digestion of the plasmid
1. set up a digestion of at least 2ug of DNA:
5uL plasmid from the miniprep
1uL enzyme
2.5uL buffer
2.5uL BSA if necessary
13.5 or 16uL water
   (25uL total)
incubate at 37C for 3h. Add 0.5uL of additional enzyme after 2h.

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):
1ug of template DNA (usually 10uL)
2uL Sp6/T7 buffer (Epicentre, in the “RNA DIG” drawer at 20C with other enzymes)
2uL DIG RNA labeling mix (Roche, in the “RNA DIG” drawer at 20C with other enzymes)
2uL 100mM DTT (stored at –20C in the in situ drawer in a green box)
2uL Sp6/T7 enzyme (in the “RNA DIG” drawer at 20C with other enzymes)
       xuL water

3. mix thoroughly and incubate at 37C for 2-2.5h (program #55).
4. add 6uL RNAse-free water (from the “RNA DIG” drawer).
5. check 1uL 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 25uL of the 2x carbonate buffer solution (=AB solution from Amikurasan, in Amikurasan drawer = 120 mM Na2CO3, 80 mM NaHCO3, pH to 10.2, 10mM DTT, aliquot and store at -20° C)
2. mix and incubate at 65C for 30min (vary from 20 to 40min to control average probe fragment size)
3. add 50uL of the stop solution(=NB solution from Amikurasan, in Amikurasan drawer = 0.3M NaOAc, 10% acetic acid, 10mM DTT)
4. add 10uL of 4M LiCl (12.08g of LiCl in 50mL total, add LiCl slowly to the water because dissolving LiCl is exothermic)
5. add 300uL ethanol 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 (for less than 1h)
10. dissolve pellet in 200 µl hybridization solution. 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.