Probe Amplification
Note: In order to have sufficient representation of the shRNA sequences from the experimental cell population, a large quantity of gDNA (20-30µg) must be used as template for probe amplification.
Note: Use plugged tips for ALL PCR setup steps. Unplugged tips may be used for all steps after amplification.
- Per sample, add the reagents below to a sterile eppendorf tube. Mix sample well after addition of water, before adding PFX (pipette 500µl up and down 8 times).
- Remove all buffers from the area of PCR set-up before opening of gDNA sample tubes.
- Add gDNA to each PCR mixture, mix samples again by pipetting 500µl up and down 10 times, then close the eppendorf tubes until samples are aliquoted into PCR tubes.
- Aliquot 99µl into each tube on a strip of 8 (54k) or 12 (80k) tubes.
PCR Recipe
54k pool 80k pool -ve control
10x PCR amplification buffer 160µl 240µl 20µl
10x enhancer buffer 160µl 240µl 20µl
10mM dNTP mix 24µl 36µl 3µl
20uM primer mix 36µl 54µl 4.5µl
50mM MgSO4 16µl 24µl 2µl
Water (392µl-Xµl gDNA) 392µl-Xµl 588µl-Xµl 49µl
PFX enzyme 12µl 18µl 1.5µl
MIX MIX MIX
gDNA template Xµl (20µg) Xµl (30µg)
MIX MIX
800µl 1200µl
Amplification Program
Step Temp Time (min)
1 94°C 5:00
2 94°C 0:15
3 55°C 0:15
4 68°C 0:20
5 Go To Step 2 (29X)
6 68°C 5:00
7 4°C hold
- After program finishes, do not store the product at either 4°C or -20°C as this enables the conversion of double-stranded product to restriction digest-resistant cruciform DNA. Instead, immediately purify the PCR-products using the QIAquick PCR purification kit and the modified protocol below.
- When run on a 2% agarose gel, the PCR product should have a pre-dominance of ~177bp product (example A below), and as little as possible of the ~225bp band. Both bands actually contain the same sequence of bases, but the larger band is composed of two DNA strands in a cruciform structure, centered around the palidromic shRNA sequence. PCR product in this form is resistant to restriction digest at the XHOI site in the middle of the shRNA palindrome since the DNA is not double stranded in this area. If the cruciform product predominates (as in example B below), try reducing the total number of PCR cycles to 29 or 28.
Probe Purification
Quantities are described for 54k or (80k) PCR purification
- Combine the 8 or (12)x99µl PCR reactions in a 15-ml screw-cap tube, and remove 10µl to run on a gel.
- Add 4 (6) ml Qiagen buffer PB to the sample and vortex to mix.
- The sample will be split onto 2 (3) columns, using half the sample per column:
- Load 750µl of sample at a time on each column and centrifuge for 60 seconds at 13,000rpm.
- Discard flow-through and repeat until all the sample has been passed through the columns.
- To wash, add 750µl Qiagen buffer PE to the column and centrifuge for 60 seconds at 13,000 rpm
- Aspirate flow-through and place the column back in the same tube.
- Centrifuge for 60 seconds at 13,000 rpm to dry the column.
- Place each column in a clean 1.5ml microcentrifuge tube.
For 54k PCR samples
- Add 30µl elution buffer EB to the centre of the membrane on each column. Let sit for 1 minute then centrifuge for 1 min at 13,000 rpm.
- Repeat elution step with another 30µl of EB and combine elutions from the 2 columns of the same tube.
- Set aside 1.5µl of the approximately 110µl total volume of purified PCR-sample to check on a gel.
For 80k PCR samples
- Add 40µl elution buffer EB to the centre of the membrane on each column. Let sit for 1 minute then centrifuge for 1 min at 13,000 rpm.
- Set aside 1µl of the approximately 110µl total volume of purified PCR-sample to check on a gel.
- Measure DNA concentration of the sample.
- When run on a 2% agarose gel, the pre and post-column purification samples should contain PCR product bands of roughly equal intensity, and the oligos should be absent in the post-column samples (see below).
Restriction Digest of PCR Product
- Set up restriction digest reaction:
Purified PCR-product 105µl
10x NEB buffer 2 12µl
10mg/ml BSA 1.2µl
XHOI (10,000U/ml ) 2µl
120.2µl
- Mix contents well by pipetting, then divide into 2x 60µl in PCR tubes.
- Incubate for 2 hours at 37°C in thermal cycler. Stop reaction by heating at 65°C for 20 min, then cooling to 4°C. Once it has been cut, it is safe to leave the sample at 4°C for extended periods if necessary.
Gel Electrophoresis and Purification of Probe
- Prepare 2% agarose gel(s) with lanes large enough to accommodate 150µl. Wide and thin lanes preferred over thick lanes so that the bands are compact, not smeared.
- Combine 2x60µl of digested sample and add loading dye, mix.
- Run the gel at 120V for 45 minutes.
- Take a quick picture of the gel for documentation, leaving the gel on the tray to minimizing UV exposure.
- Using fresh razor blades, cut out the probe for each sample. The brightest, middle band, running at ~100bp contains the desired probe DNA. Trim as much excess agarose around the band as possible.
- Transfer the gel slices to pre-weighed 15ml screw-cap tubes .
- Weigh the tubes containing gel slices and calculate the weight of the gel slices (should be between 300-400mg).
- Add 3 gel volumes of buffer QG (eg. 300µl per 100mg).
- Incubate at 50°C for 15-20 minutes, vortexing every 2-3 minutes.
- To ensure complete dissolution of the agarose, the samples should remain in the water bath for several minutes after it has visibly dissolved.
- Add 1 gel volume of isopropanol to the sample and vortex.
- 400mg is the maximum gel weight per column, divide samples if your slice(s) weighed more.
- Add 750µl sample to each column and centrifuge 1 minute at 13,000rpm.
- Discard flow-through and repeat until all the sample has been passed through the columns.
- Add 750µl buffer PE. Incubate for 5 minutes at room temperature, then centrifuge 1 minute at 13,000rpm.
- Aspirate the flow-through to remove all of the PE buffer.
- Centrifuge tubes at 13,000 rpm for 1 minute, to dry the column.
- Put columns in clean 1.5ml tubes.
- Add 30µl EB to each column, leave at room temperature for 1 minute then centrifuge for 1 minute at 13,000rpm.
- Repeat elution step with another 30µl of EB and combine elutions from the 2 columns of the same sample (~60µl).
- Check the concentration and purity on a spectrophotometer.
- Expect the 260/280 ratio to be 1.8-2.0, and the 260/230 to be 0.2-0.4.
- Use the Qiagen PCR purification kit to clean the samples and remove more of the salts (remaining from the QG buffer).
- Use one column per sample, do one PE wash with a 5 minute incubation prior to centrifuging.
- Elute 2x30µl with EB, incubating for 1minute at RT before centrifuging.
- Check the concentration and purity on a spectrophotometer.
- Expect the 260/280 ratio to be 1.8-2.0, and the 260/230 to be 1.7-2.1.
- You should have over 2µg (average yield is 3-3.5µg) of each sample to hybridize on chips.
Pre-conditioning of chips
- Let chips warm up to room temperature on bench for about an hour.
- Fill the chip slowly with 40°C 10mM NaOH, then remove the liquid.
- Fill the chip again with 40°C 10mM NaOH. Incubate with rotation in hybridization oven for 10 minutes at 40°C, 40rpm.
- Cut the head off of a 200µl pipette tip and fit the tip onto a 5ml-syringe.
- Rinse chip slowly with 3-5mL wash buffer A using the 5ml syringe, collecting the flow-through in a beaker.
- Remove wash Buffer A, then fill the array with 0.0005% Triton and incubate in hybridization oven for 10 min at 40°C, 40rpm.
- Empty the chip, then rinse it 2x with wash buffer A by filling and emptying the chip.
- Fill up chip with wash buffer A.
- Put chips in the hybridization oven at 40°C, 40-60 rpm for 2-4hrs.
Hybridization Mix preparation
- The mixture of blocking oligos is made up of block_1, block_2, block_3 and block_4, each at a concentration of 20µM.
- Aliquots of ~105µl (4 chips worth) should be prepared to reduce the number of freeze/thaw cycles on stocks.
- Prepare the probe mixture in an eppendorf:
2x hybridization buffer 150µl
50mg/ml BSA 3µl
10mg/ml herring sperm DNA 3µl
5nM B213 2.9µl
Blocking oligo mix 25µl
DMSO 30µl
Sample (2µg) Xµl
Water 100µl-Xµl
213.9µl
- Incubate the tube in a boiling water bath for 10 minutes.
- Transfer sample to a 40°C water bath for 5 minutes.
- Centrifuge tubes at 13,000 rpm for 5 minutes.
- Remove wash A from the chips and fill them with the sample solution, avoiding any debris in the bottom of the tube.
- Cover the ports on the chips with tough-spot seals.
- Incubate the chips for 15-16 hours at 40°C, 60 rpm.
Chip staining and washing
Prime the fluidics station:
- Put all tubing in place, empty waste bottle, fill wash A and B bottles, fill up Millipore water bottle.
- Use fluidics station protocol “PRIME_450”.
- Prepare SAPE labeling mix and make 2 aliquots of 590µl each per chip.
- Prepare antibody labeling mix and make 1 aliquot of 590µl per chip.
SAPE labeling mix per chip:
2x MES stain buffer 600µl
50 mg/ml BSA 48µl
1 mg/ml SAPE 12µl
High quality water 540µl
1200µl
Antibody labeling mix per chip:
2x MES stain buffer 300µl
50 mg/ml BSA 24µl
30 mg/ml IgG 2µl
500 µg/ml BAS* 3.6µl
High quality water 270.4µl
600µl
- Aliquot 590µl/chip for each solution into eppendorf tubes.
- Remove tough-spots from chips .
- Remove hybridization mix (~2x150µl) and set aside for -20°C storage for potential future use.
- Fill chip with wash A.
- On scanner computer, under “Experiments”, scan the barcode and type in the sample information.
- Use the protocol “FlexGE_WS2v5_450_KCEE” (runtime is ~75 min).
- When the chips are ready, check for air bubbles and wash again if necessary.
- Put tough spots on the chips and scan them.
- When done with the fluidics, put all tubing in Millipore water and run “SHUTDOWN_450” program.
- When shutdown and scanning is finished, release all fluidics tubing and shut off stations and laser.