{"id":6518,"date":"2016-09-09T13:54:53","date_gmt":"2016-09-09T05:54:53","guid":{"rendered":"http:\/\/biopioneer.com.tw\/?p=6518"},"modified":"2025-08-27T18:39:06","modified_gmt":"2025-08-27T10:39:06","slug":"%e5%b0%be%e5%b7%b4gdna%e8%90%83%e5%8f%96-directpcr-lysis-reagent%e8%b2%a8%e8%99%9f-101-t","status":"publish","type":"post","link":"http:\/\/biopioneer.com.tw\/?p=6518","title":{"rendered":"\u5feb\u901f\u5c3e\u5df4gDNA\u8403\u53d6 (Tail) |DirectPCR Lysis Reagent\u8ca8\u865f 101-T\/102-T"},"content":{"rendered":"

\u57fa\u56e0\u578b\u9451\u5b9a\u7522\u54c1\u4ecb\u7d39 |\u00a0<\/span>\u6700\u7c21\u55aegDNA\u8403\u53d6\u65b9\u6cd5-\u5feb\u901f\/\u514dColumn\/\u55ae\u7ba1\u64cd\u4f5c\u00a0\u00a0<\/span><\/span><\/p>\n

Genotyping without DNA isolation<\/p>\n

\"\u54c1\u724c\u5546\u54c1\u7e6a\u5716<\/p>\n

DirectPCR Lysis Reagent (mouse tail),\u00a0 Cat#102-T ( 100m l\u5305\u88dd)<\/a><\/span><\/p>\n

\"viagenbiotech\"<\/p>\n

\u50b3\u7d71\u8403\u53d6 \u201c\u5c3e\u5df4gDNA\u201d\uff0c\u7121\u6cd5\u76f4\u63a5PCR\uff1b\u56e0\u7d44\u7e54\u5167\u542b\u6709PCR\u53cd\u61c9\u6291\u5236\u56e0\u5b50\uff0c\u6291\u5236PCR\u53cd\u61c9\u3002\u9808\u7d93\u7531\u6709\u6a5f\u6eb6\u5291\u6216kit\u8403\u53d6gDNA\u3002\u201c<\/em><\/strong>\u5c08\u5229\u7814\u767c\u201d <\/em><\/strong>\u5177\u6709\u6291\u5236PCR\u6291\u5236\u7269\u7684\u53cd\u61c9\uff0c\u4e0d\u9808\u8003\u91cfPCR\u6291\u5236\u554f\u984c\u3002DirectPCR Lysis Reagent (mouse tail), Cat# 101-T( 50m l\u5305\u88dd), Cat#102-T( 100m l\u5305\u88dd)\uff0c\u53ea\u8981\u4e00\u500b\u52a0\u71b1\u6b65\u9a5f\u53cd\u61c9<\/strong>\uff0c\u5373\u53ef\u5c07DNA\u7531\u7d44\u7e54\u4e2d\u91cb\u653e\uff0c\u4e26\u4e14\u4e0d\u9808\u7d93\u7531\u4efb\u4f55\u7d14\u5316\u7684\u6b65\u9a5f, \u4f8b\u5982Geneaid genomicDNA purification\u6216\u6709\u6a5f\u6eb6\u5291Phenol\/Chloroform\u7d14\u5316\uff0c\u52a0\u71b1\u5b8c\u5f8c\u7684\u6eb6\u6db2\u53ef\u4ee5\u76f4\u63a5PCR\uff0c\u9032\u884cgenotyping\u5206\u6790\u3002<\/p>\n

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.<\/p>\n

\"\"<\/p>\n

\u6700\u7d93\u6fdf\u7684\u8403\u53d6\u8a66\u5291<\/strong>\u00a0500\u6b21\u5c3e\u5df4 ( 0.5c m\u5c3e\u5df4\u52a0\u5165200ul\u70ba\u4f8b )\u3002<\/p>\n

Brief procedure:<\/strong>
\n1. \u00a0Lyse tails in DirectPCR\u00ae Lysis Reagent.
\n2. \u00a0Incubate for 45 min at 85\u00b0C.
\n3. \u00a0PCR genotyping with 1 \u00b5l lysates.<\/p>\n

\u64cd\u4f5c\u624b\u518a (\u4f9d\u539f\u5ee0\u7db2\u7ad9\u70ba\u4e3b)<\/p>\n

1. For 0.5 cm tail, add 200\u2013300 \u00b5l 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 \u00b5l Proteinase K solution per 1 ml DirectPCR reagent). See Table 1 for starting conditions.<\/p>\n

NOTE: Although 200 \u00b5l DirectPCR is usually sufficient for complete lysis of 0.5 cm tail, application of 250-300 \u00b5l 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.<\/p>\n

\"102-t\"<\/p>\n

 <\/p>\n

2. Rotate the tubes in rotating hybridization oven at 55\u00b0C 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.<\/p>\n

NOTE: Rotating hybridization oven performs better than rocking plate. Use 0.75 cm tubes for less than 200 \u00b5l 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.<\/p>\n

3. Incubate crude lysates at 85\u00b0C 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\u00b0C for 1 year (or at 4\u00b0C for 1 week) without losing efficacy.<\/p>\n

4. Use 0.5-1.0 \u00b5l of lysate for 50 \u00b5l 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.<\/p>\n

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\u00b0C for 2 min, discard supernatant, wash DNA with 1 ml 70% EtOH, and dissolve DNA in 50 \u00b5l 10 mM Tris-HCl (8.0). Use 1 \u00b5l for PCR.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
DirectPCR DNA Extraction Reagents<\/strong><\/td>\nCat#<\/strong><\/td>\nComments<\/strong><\/td>\n<\/tr>\n
DirectPCR Lysis Reagent (mouse tail)<\/td>\n[101-T]<\/td>\n250 mouse tails (50 ml)<\/td>\n<\/tr>\n
DirectPCR Lysis Reagent (mouse tail)<\/td>\n[102-T]<\/td>\n500 mouse tails (100 ml)<\/td>\n<\/tr>\n
DirectPCR Lysis Reagent (yolk sac)<\/td>\n[201-Y]<\/td>\nyolk sacs (50 ml)<\/td>\n<\/tr>\n
DirectPCR Lysis Reagent (yolk sac)<\/td>\n[202-Y]<\/td>\nYolk sacs (100 ml)<\/td>\n<\/tr>\n
DirectPCR Lysis Reagent (cell<\/td>\n[301-C]<\/td>\ncultured cells (50 ml)<\/td>\n<\/tr>\n
DirectPCR Lysis Reagent (cell)<\/td>\n[302-C]<\/td>\ncultured cells (100 ml)<\/td>\n<\/tr>\n
DirectPCR Lysis Reagent (mouse ear)<\/td>\n[401-E]<\/td>\nmouse ear (25 ml)<\/td>\n<\/tr>\n
DirectPCR Lysis Reagent (mouse ear)<\/td>\n[402-E]<\/td>\nmouse ear (50 ml)<\/td>\n<\/tr>\n
<\/td>\n<\/td>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

VIAGEN BIOTECH\u7522\u54c1\u5982\u4f55\u5206\u985e?<\/strong><\/p>\n

VIAGEN BIOTECH\u5c07\u7522\u54c1\u5206\u70ba\u56db\u7a2e\uff0c\u8996\u5ba2\u6236\u9700\u6c42\u9078\u64c7\u4e0d\u540c\u7522\u54c1: \u5176\u5206\u985e\u6709\u5c3e\u5df4genomic DNA\u8403\u53d6\u3001\u7d30\u80degenomic DNA\u8403\u53d6\u3001\u8033\u6735genomic DNA\u8403\u53d6\u3001\u5375\u9ec3\u56cagenomic DNA\u8403\u53d6\u3002<\/p>\n

 <\/p>\n

\u50b3\u7d71\u5c3e\u5df4gDNA\u8403\u53d6, \u90fd\u9808\u7d93\u7531Phenol\/Chloroform\u8403\u53d6\u6b65\u9a5f, \u6216\u8005\u5e02\u552egenomic DNA Purification\u5957\u7d44(\u901a\u904ecolumn\u7b49\u7e41\u7463\u6b65\u9a5f)\u4f86\u7d14\u5316genomic DNA, \u70ba\u4f55VIAGEN BIOTECH\u516c\u53f8\u51fa\u552e\u7684\u7522\u54c1, \u53ef\u4ee5\u4e00\u500b\u6b65\u9a5f\u5b8c\u6210\u53cd\u61c9?<\/p>\n

\u50b3\u7d71\u8403\u53d6 \u201c\u5c3e\u5df4gDNA\u201d\uff0c\u7121\u6cd5\u76f4\u63a5PCR\uff1b\u56e0\u7d44\u7e54\u5167\u542b\u6709PCR\u53cd\u61c9\u6291\u5236\u56e0\u5b50\uff0c\u6291\u5236PCR\u53cd\u61c9\u3002\u9808\u7d93\u7531\u6709\u6a5f\u6eb6\u5291\u6216kit\u8403\u53d6gDNA\u3002\u201c<\/em><\/strong>\u5c08\u5229\u7814\u767c\u201d <\/em><\/strong>DirectPCR Lysis Reagents\u5177\u6709\u6291\u5236PCR\u6291\u5236\u7269\u7684\u53cd\u61c9\uff0c\u4e0d\u9808\u8003\u91cfPCR\u6291\u5236\u554f\u984c\u3002VIAGEN BIOTECH\u516c\u53f8\u5c08\u9580\u4ee5DirectPCR Lysis Reagent\u92b7\u552e\u70ba\u4e8b\u696d\uff0c \u5176\u516c\u53f8\u7814\u767c\u3001\u751f\u7522\u3001\u92b7\u552e\u3002\u54c1\u8cea\u7a69\u5b9a\uff0c\u92b7\u552e\u7b2c\u4e00\u3002<\/p>\n

 <\/p>\n

genomic DNA\u6c92\u6709\u7d93\u7531\u8403\u53d6\u6b65\u9a5f,\u4e0d\u77e5genomic DNA\u54c1\u8cea\u5982\u4f55?genomic DNA\u7684\u7a69\u5b9a\u6027\u5982\u4f55?<\/p>\n

VIAGEN BIOTECH\u5c08\u9580\u70bagenomic DNA\u8403\u53d6\u800c\u8a2d\u8a08\uff0cPCR\u53cd\u61c9\u5f8c\u7522\u7269, \u53ef\u4ee5\u5927\u65bc15Kb, \u5c3e\u5df4\u7d93\u7531DirectPCR Lysis Reagent\u5f8c, \u5176\u57284\u5ea6C\uff0c\u53ef\u4ee5\u7dad\u6301\u4e0d\u8b8a\u7684\u6548\u80fd\u3002\u5728-20\u5ea6C, \u53ef\u4ee5\u7dad\u63011\u5e74\u54c1\u8cea\u90fd\u4e0d\u6703\u6539\u8b8a\u3002<\/p>\n

 <\/p>\n

\u5c3e\u5df4\u7d93\u7531DirectPCR Lysis Reagent\u5f8c\uff0c\u5982\u4f55\u7d14\u5316Crude lysates?<\/p>\n

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\u00b0C for 2 min, discard supernatant, wash DNA with 1 ml 70% EtOH, and dissolve DNA in 50 \u03bcl 10 mM Tris-HCl (8.0). Use 1 \u03bcl for PCR.<\/p>\n

 <\/p>\n

\u9700\u8981\u6dfb\u52a0Proteinase K\u55ce?<\/p>\n

\u9700\u8981\uff0cProteinase K 0.2 -0.4 m g\/ml\u00a0\u00a0\u86cb\u767d\u9176 K (Proteinase K) | CAS#39450-01-6 APExBIO\u00a0 \u8ca8\u865fK1037<\/a><\/p>\n

\"\u76d2\u5b50\u6a23\u54c12\"<\/p>\n

 <\/p>\n

Important Technical Tips<\/p>\n

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,<\/p>\n

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.<\/p>\n

3. Taq polymerase. We have tested many types of commercially available Taq polymerases. The listed enzymes are recommended for optimal results.<\/p>\n

4. Tissue size. The size of tails should be 0.5 cm or slightly smaller. Use a minimal volume (0.5-1 \u00b5l for 50 \u00b5l PCR reaction) of crude lysates for PCR amplification. Too much DirectPCR reagents inhibit PCR efficiency.<\/p>\n

5. Small tubes and evaporation. To minimize evaporation, use a 0.75 ml tube when the reagent volume is less than 100 \u00b5l.<\/p>\n

6. Small samples and dilution. If the required DirectPCR reagent volume is less than 50 \u00b5l, 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.<\/p>\n

7. PCR machine. PCR machines are occasionally a source of technical problem<\/p>\n

\"\u64cd\u4f5c\u624b\u518a2\"<\/a><\/p>\n","protected":false},"excerpt":{"rendered":"

\u57fa\u56e0\u578b\u9451\u5b9a\u7522\u54c1\u4ecb\u7d39 |\u00a0\u6700\u7c21\u55aegDNA\u8403\u53d6\u65b9\u6cd5-\u5feb\u901f\/\u514dColumn\/\u55ae\u7ba1\u64cd\u4f5c\u00a0\u00a0 Genotyping wi […]<\/p>\n","protected":false},"author":3,"featured_media":37026,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":[],"categories":[88],"tags":[4493],"_links":{"self":[{"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/posts\/6518"}],"collection":[{"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=6518"}],"version-history":[{"count":4,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/posts\/6518\/revisions"}],"predecessor-version":[{"id":52462,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/posts\/6518\/revisions\/52462"}],"wp:featuredmedia":[{"embeddable":true,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/media\/37026"}],"wp:attachment":[{"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=6518"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=6518"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=6518"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}