基因型鑑定產品介紹 | 最簡單gDNA萃取方法-快速/免Column/單管操作

Genotyping without DNA isolation

DirectPCR Lysis Reagent (mouse tail),  Cat#102-T ( 100m l包裝)

傳統萃取 “尾巴gDNA”，無法直接PCR；因組織內含有PCR反應抑制因子，抑制PCR反應。須經由有機溶劑或kit萃取gDNA。“專利研發” 具有抑制PCR抑制物的反應，不須考量PCR抑制問題。DirectPCR Lysis Reagent (mouse tail), Cat# 101-T( 50m l包裝), Cat#102-T( 100m l包裝)，只要一個加熱步驟反應，即可將DNA由組織中釋放，並且不須經由任何純化的步驟, 例如Geneaid genomicDNA purification或有機溶劑Phenol/Chloroform純化，加熱完後的溶液可以直接PCR，進行genotyping分析。

DirectPCR Lysis Reagent (Mouse Tail) 100ml (Up to 500 Tails). The system is a single-tube system for rapid preparation of DNA from mouse tails. The patent-pending components allow the resulting DNA extracts to be compatible with genomic PCR for genotyping. Crude extracts of biological samples are not compatible with many molecular biology-grade reactions such as polymerase chain reaction (PCR), in part due to inhibitors contained in crude extracts. The DirectPCR reagents not only mediate the rapid lysis of biological samples but also contain inhibitors that effectively suppress the inhibitory activities of crude lysates for PCR amplification, while maximally maintaining the integrity of released genomic DNA. The patent-pending simple procedure completely eliminates any solution transfer or tube-opening steps, providing you with substantial extra time. Stable for 12 months at 4Deg C / Ships at room temperature.

最經濟的萃取試劑 500次尾巴 ( 0.5c m尾巴加入200ul為例 )。

Brief procedure:
1.  Lyse tails in DirectPCR® Lysis Reagent.
2.  Incubate for 45 min at 85°C.
3.  PCR genotyping with 1 µl lysates.

操作手冊 (依原廠網站為主)

1. For 0.5 cm tail, add 200–300 µl DirectPCR Lysis Reagent (Tail) containing freshly prepared 0.2-0.4 mg/ml Proteinase K (Sigma, cat # p6556, not included). Proteinase K is stable in DirectPCR reagents for ~24 hrs. If a small number of tails are processed, and therefore it is difficult to weigh Proteinase K powder, use genomic PCR-quality Proteinase K solution (Viagen, cat #501-PK) at 0.5-1.0 mg/ml (25-50 µl Proteinase K solution per 1 ml DirectPCR reagent). See Table 1 for starting conditions.

NOTE: Although 200 µl DirectPCR is usually sufficient for complete lysis of 0.5 cm tail, application of 250-300 µl yields more reproducible results because of better mixing efficiency. Compare several different volumes of DirectPCR reagents for best performance. If tails are not mixed well with solutions, use 0.75 ml tubes.

2. Rotate the tubes in rotating hybridization oven at 55°C for 5-6 hrs or until no tissue clumps are observed. If necessary, rotation can be allowed overnight without loss of efficacy. Complete lysis is important. Since some tails may not be in contact with solutions, re-position once the tails by shaking the bottles containing tubes, preferentially after 2-3 hrs.

NOTE: Rotating hybridization oven performs better than rocking plate. Use 0.75 cm tubes for less than 200 µl of DirectPCR Reagent. DNA fragmentation by prolonged rotation will not influence significantly PCR performance. Use roughly proportional volume of DirectPCR Lysis Reagent for different sized samples.

3. Incubate crude lysates at 85°C for 45 min by floating the whole rack (containing tubes) on a water bath. (Optional) Precipitate hairs by centrifuging for 10 sec before step 4. Crude lysates may be stored at -20°C for 1 year (or at 4°C for 1 week) without losing efficacy.

4. Use 0.5-1.0 µl of lysate for 50 µl PCR reaction. Eppendorf Hotmaster Taq Polymerase (cat# 954-14-5018), Sigma JumpStart Taq DNA Polymerase (cat# D9307), or Qiagen HotStar Taq DNA polymerase (cat# 203203) is recommended for PCR.

Rescue of DNA: DNA in crude lysates can be rescued for further analysis. Add NaCl to a final concentration of 250 mM, and then add 0.7 volume of isopropanol. DNA will form precipitates. Centrifuge at 4°C for 2 min, discard supernatant, wash DNA with 1 ml 70% EtOH, and dissolve DNA in 50 µl 10 mM Tris-HCl (8.0). Use 1 µl for PCR.

DirectPCR DNA Extraction Reagents	Cat#	Comments
DirectPCR Lysis Reagent (mouse tail)	[101-T]	250 mouse tails (50 ml)
DirectPCR Lysis Reagent (mouse tail)	[102-T]	500 mouse tails (100 ml)
DirectPCR Lysis Reagent (yolk sac)	[201-Y]	yolk sacs (50 ml)
DirectPCR Lysis Reagent (yolk sac)	[202-Y]	Yolk sacs (100 ml)
DirectPCR Lysis Reagent (cell	[301-C]	cultured cells (50 ml)
DirectPCR Lysis Reagent (cell)	[302-C]	cultured cells (100 ml)
DirectPCR Lysis Reagent (mouse ear)	[401-E]	mouse ear (25 ml)
DirectPCR Lysis Reagent (mouse ear)	[402-E]	mouse ear (50 ml)

【產品問與答】VIAGEN BIOTECH產品如何分類?

VIAGEN BIOTECH將產品分為四種，視客戶需求選擇不同產品: 其分類有尾巴genomic DNA萃取、細胞genomic DNA萃取、耳朵genomic DNA萃取、卵黃囊genomic DNA萃取。

【產品問與答】傳統尾巴gDNA萃取, 都須經由Phenol/Chloroform萃取步驟, 或者市售genomic DNA Purification套組(通過column等繁瑣步驟)來純化genomic DNA, 為何VIAGEN BIOTECH公司出售的產品, 可以一個步驟完成反應?

傳統萃取 “尾巴gDNA”，無法直接PCR；因組織內含有PCR反應抑制因子，抑制PCR反應。須經由有機溶劑或kit萃取gDNA。“專利研發” DirectPCR Lysis Reagents具有抑制PCR抑制物的反應，不須考量PCR抑制問題。VIAGEN BIOTECH公司專門以DirectPCR Lysis Reagent銷售為事業， 其公司研發、生產、銷售。品質穩定，銷售第一。

【產品問與答】genomic DNA沒有經由萃取步驟,不知genomic DNA品質如何?genomic DNA的穩定性如何?

VIAGEN BIOTECH專門為genomic DNA萃取而設計，PCR反應後產物, 可以大於15Kb, 尾巴經由DirectPCR Lysis Reagent後, 其在4度C，可以維持不變的效能。在-20度C, 可以維持1年品質都不會改變。

【產品問與答】尾巴經由DirectPCR Lysis Reagent後，如何純化Crude lysates?

DNA in crude lysates can be rescued for further analysis. Add NaCl to a final concentration of 250mM , and then add 0.7 volume of isopropanol. DNA will form precipitates. Centrifuge at 4°C for 2 min, discard supernatant, wash DNA with 1 ml 70% EtOH, and dissolve DNA in 50 μl 10 mM Tris-HCl (8.0). Use 1 μl for PCR.

【產品問與答】需要添加Proteinase K嗎?

需要，Proteinase K 0.2 -0.4 m g/ml

Important Technical Tips

1. Complete lysis. Big tissue clumps should not be observed after digestion. It is recommended to vigorously shake the bottle (containing microfuge tubes) for 2-3 sec anytime, once or twice, after tissues begin to partially dissolve. This will physically disperse partially digested tissues and reposition microfuge tube, in which tails are separated from lysis reagents, thereby facilitating overall lysis efficiency,

2. Proteinase K inactivation. Inactivation of proteinase K by incubating samples at 85C-86C for 45-50 min is critical to protect Taq polymerase from proteinase K.

3. Taq polymerase. We have tested many types of commercially available Taq polymerases. The listed enzymes are recommended for optimal results.

4. Tissue size. The size of tails should be 0.5 cm or slightly smaller. Use a minimal volume (0.5-1 µl for 50 µl PCR reaction) of crude lysates for PCR amplification. Too much DirectPCR reagents inhibit PCR efficiency.

5. Small tubes and evaporation. To minimize evaporation, use a 0.75 ml tube when the reagent volume is less than 100 µl.

6. Small samples and dilution. If the required DirectPCR reagent volume is less than 50 µl, dilute the reagent by up to 2-fold with water, while maintaining the same concentration of proteinase K. If the DirectPCR reagent is ‘2- fold’ diluted, apply ‘2-fold’ more crude lysates for PCR reaction.

7. PCR machine. PCR machines are occasionally a source of technical problem