1.5.1 Lysis of the cells:
Prepare a stock of a 1x direct lysis buffer (1 mM CaCl2, 3 mM MgCl2, 1 mM EDTA, 1% Triton X-100, 10 mM Tris pH 7.5)
Add 0.2 mg/ml Proteinase K to an aliquot of the lysis buffer
Remove the medium from one copy of the cell clones
Add 30�l lysis buffer and resuspend, transfer to a 96-well PCR plate
Incubate for 10min at 65C and for 15min at 95C, then store on ice
1.5.3 Preparation of barcode primer plate:
Pipet 2.5�l of each of the primers D501-D508 into all wells of rows A-H, respectively, of a round-well 96-well plate
Pipet 2.5�l of each of the primers D701-D712 into all wells of column 1-12, respectively, of the plate
Add 95�l water to each well of the plate and resuspend
1.5.4 Second PCR
Prepare a 120x PCR mastermix containing:
- 300�l Phusion HF buffer
- 30�l dNTP mix 10mM
- 15�l Phusion polymerase
- 615�l water
In a 96-well PCR plate, mix 2�l first PCR, 2.5�l barcode primer mix, and 8�l mastermix. Avoid contaminating the barcode plate.
Run with the protocol: 2min 98C, 19x(20sec 98C, 20sec 60C, 30sec 72C), 2min 72C
1.5.5 Prepare DNA for a MiSeq run:
Mix all PCR products in a single tube
Run PCR products on a 1% agarose gel, cut the 400-500bp band, purify it using a column purification kit, and elute in 200�l water
Precipitate by adding 1�l glycogen, 20�l 3M NaOAc pH 5.2, 220�l isopropanol, incubate for 20minutes on ice
Spin for 15 minutes at 4C, remove supernatant, wash with 70% ethanol, spin for 5 minutes, remove most liquid
Pulse spin, remove remaining liquid, dry pellet until it gets transparent
Resuspend in 35�l water for 5min at 37C, spin 3min full speed, carefully transfer 25�l from the top to a fresh tube
Measure the concentration twice using a NanoDrop spectrometer
1.5.6 Start a MiSeq run
Thaw a MiSeq 300 cycles v2 cassette in a waterbath for 1h
Rename
this samplesheet to the name of the cassette (typically MS...-300V2) and copy it to the folder D:\Illumina\...
Mix appropriate amounts of all DNA samples to be sequenced into 350�l of Illumina hyb buffer. 32ng of DNA roughly give rise to 1 mio reads. For genotyping 96 clones, anything above 200,000 reads would be sufficient.
Mix 10�l of DNA dilution with 10�l 0.2N NaOH, incubate for 5 minutes at room temperature, then add 980�l hyb buffer and store on ice
Mix 180�l of denatured DNA with 420�l of hyb buffer in a fresh tube and transfer completely to the sample well of the reagent cassette. Store cassette on ice.
Clean flow cell with water and ethanol and insert into the MiSeq. Insert cassette and buffer flask, empty the waste container and start the standard sequencing program.
1.5.7 Retrieve and analyze the data:
When the run is finished after about 22h, follow the wash instructions on the MiSeq screen
Copy the 384 zipped FASTQ files from the folder D:\Illumina\Miseq output\-your-run-\data\intensities\basecalling to a USB drive
Copy the files to your computer and unzip them (double-click on a mac), then delete the zipped files
Click ANALYZE DATA below to analyze sequencing data and follow the instructions