Abi 3730xl automatic sequencer

Genomic DNA was extracted from frozen BM mononuclear cells from two patients and from the unstained BM slides of 14 patients. DNA was extracted using the MagNA Pure LC DNA Isolation Kit (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer's instructions. The quality of DNA was analyzed by assessing the 260/280 absorbance ratio using an ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).
Two primers (forward, 5′-CTG GCA AGA GAA TGA GGG AAT GT-3′; reverse, 5′-AGG AGG CAG GGC AGA AGT A-3′) were used to amplify a 489-base pair fragment covering the MYD88 L265P site. PCR was performed using 25 ng to 100 ng genomic DNA in 100 μL of PCR solution (10 μL of 10× MG Taq-HF buffer, 0.2 μM of each primer, 10 μL of 2 mM MG dNTPs mixture, 1 μL of MG Taq-HF polymerase (Macrogen Inc., Seoul, Korea), and distilled water). PCR was performed using an initial denaturation step of 5 min at 94°C, followed by 35 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 60 s, with a final extension of 7 min at 72°C. The PCR products were purified and sequenced using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) and an ABI 3730 XL automatic sequencer (Applied Biosystems) using the same primers described above.

To obtain single PoMaf1 KO cells, HINAE cells were transfected with Cas9/PoMaf1 sgRNA and EGFP co-expression vector (pSpCas9(BB)-2A-GFP-PoMaf1 sgRNA). At 48 h post transfection, cells were trypsinized for 3 min at room temperature and collected by centrifugation. Cells were resuspended in L-15 culture medium containing 15% heat-inactivated FBS and 1.5% (v/v) AA before being passed through a 35 μm cell strainer into tubes (BD Falcon, Franklin Lakes, NJ, USA). Cells positive for GFP (and therefore Cas9) expression were sorted and transferred to 96-well plates using a cell sorter (BD FACS Aria III; BD Biosciences, San Jose, CA, USA) following the manufacturer’s instructions. Control cells were transfected with the empty pX458 construct. After culture for one month, genomic DNA was extracted from the clones using PrimePrep Direct PCR Reagent (GeNetBio, Daejeon, Korea) following the manufacturer’s instructions, and the fragments containing the CRISPR target site were amplified by PCR using specific primers (PoMaf1-seq-F, 5′-TGT TTT GCA AGG TGA CTG TAC GT-3′; PoMaf1-seq-R, 5′-GGC GTG ACT TTT GTT GAG TAT TAA CT-3′). The PCR amplicons were purified and cloned into the pGEM^®-T easy vector (Promega), and at least eight colonies per clone were sequenced to detect indels using an ABI3730xl Automatic Sequencer (Applied Biosystems, Inc.).

The genomic DNA of the mosquitoes was individually extracted using the Insect DNA Kit (Omega Bio-tek, Norcross, GA, USA), following the manufacturer’s protocol. The quality and concentration of the extracted DNA were evaluated with a NanoDrop™ 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Extracted DNA was stored at −20 °C or used immediately for PCR. Partial domain III of VGSC gene (containing F1534 and I1532) was amplified using forward primer aegSCF7 (5′-GAG AAC TCG CCG ATG AAC TT-3′) and reverse primer aegSCR8 (5′-TAG CTT TCA GCG GCT TCT TC-3′) [19 (link)]. The PCR reaction mixture consisted of 100 ng genomic DNA, 15 μl 2 × PCR Master Mix (Promega, Madison, WI, USA), 1 μl (10 μM) forward and reverse primers, and ddH₂O, in a final volume of 30 μl. The PCR cycling conditions were as follows: 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 40 s, with a final extension at 72 °C for 10 min. The quality of PCR products was ascertained by 1.5% agarose gel electrophoresis following ethidium bromide stain. The PCR product was purified using a gel extraction kit (Omega Bio-tek) and sequenced directly with aegSCR8 using the ABI 3730XL automatic sequencer (Applied Biosystems, Guangzhou, China).

Whole-genome DNA was extracted from yeast cells using the Yeast Genomic DNA Extraction Kit (Solarbio Life Sciences, Beijing, China). The primers used for amplification and sequencing of six genes have been described previously (Tavanti et al., 2005 (link)). Amplification reactions were performed in 25 μl volume containing 12.5 μl 2^∗Taq PCR PreMix (Innovagene Biotechnology, Changsha, China), 8.5 μl double-distilled H2O, 1.5 μl forward/reverse primers, and 2.5 μl template DNA. Reaction conditions were as follows: 1 cycle of denaturation for 7 min at 94°C, followed by 30 cycles of 94°C for 1 min, 53°C for 1 min and 72°C for 1 min 5 s, with a final extension step of 10 min at 72°C. Following PCR, each product was purified with the PCR Purification Kit (Omega bio-tek, Norcross, America). Bidirectional DNA sequencing was performed by an ABI 3730XL automatic sequencer (Applied Biosystems, Foster, America). Sequencing results were spliced with Geneious 4.8 software² and polymorphic sites were confirmed by visual observation. The heterozygous sites in the chromatograms were defined by the heterozygous data (K,M,R,S,W, and Y) from the International Union of Pure and Applied Chemistry (IUPAC)³ nomenclature.

Positive PCR reactions were purified with an Illustra ExoProStar one-step kit (GE Healthcare, Chicago, IL, USA) following the manufacturer’s protocol. After PCR purification, both strands were sequenced in an ABI 3730 xl automatic sequencer (Applied Biosystems, Waltham, MA, USA).