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RT-6锛圧T-6锛変汉鐨扮柟鐥呮瘨6鍨嬫帰閲濇硶鐔掑厜瀹氶噺PCR瑭﹀姂鐩掕鏄庢浉
| 璩囨枡椤炲瀷 | doc鏂囦欢 | 璩囨枡澶у皬 | 27136 |
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RT-6(RT-6)浜虹毎鐤圭梾姣�6鍨嬫帰閲濇硶鐔掑厜瀹氶噺PCR瑭﹀姂鐩掕鏄庢浉
1銆� 瑭﹀姂鐩掔啊浠嬭波鐐轰簡閬╂噳(y墨ng)RT-6(RT-6)浜虹毎鐤圭梾姣�6鍨嬫帰閲濇硶鐔掑厜瀹氶噺PCR瑭﹀姂鐩掑揩閫熸娓拰鐤梾鐮旂┒鐨勯渶瑕侊紝鏈叕鍙稿弮鐓� OIE 妯�(bi膩o)婧�(zh菙n)涓(gu墨)瀹氱殑寮曠墿搴忓垪锛岀稉(j墨ng)澶氭瀵﹂鍙婄郴绲�(t菕ng)鍎�(y艒u)鍖栵紝闁嬬櫦(f膩)鐢熺敘(ch菐n)浜嗘湰瑭﹀姂鐩掋�傛噳(y墨ng)鐢ㄦ湰瑭﹀姂鐩掗��(j矛n)琛屾娓叿鏈夊揩閫熴�侀潏鏁忋�佺壒鐣般�佹簴(zh菙n)纰恒�佸畨鍏ㄦ搷浣滅啊鍠�佹噳(y墨ng)鐢ㄥ唬娉涘拰楂橀�氶噺妾㈡脯绛夌壒榛炲強鍎�(y艒u)榛炪��2銆佽│鍔戠洅绲勬垚:
瑭﹀姂鐩掔祫鎴愬寘鎷牳閰告彁鍙栬│鍔戝拰鏍搁吀鎿村瑭﹀姂锛屽叿楂旂祫鎴愬弮瑕嬭〃 1锛�
琛� 1锛氳│鍔戠洅绲勬垚锛�50test/鐩掞級
*淇濆瓨姊濅欢锛氭ǎ鍝� DNA 鎻愬彇娑� 1銆�2 鍜岃│鍔戠洅闋堝湪-20鈩冧繚瀛樸��2銆佹ǎ鏈噰闆嗭紝瀛樻斁鍙婇亱杓�
2.1 妯ｆ湰閲囬泦
鎵�鐢ㄥ彇妯ｅ櫒鏉愬繀闋堢稉(j墨ng)閬庨珮澹撴粎鑿屽苟鐑樺共銆傚彇灏嶈潶鐨勮叜绲层�佽倽鑳拌吅銆佽吂鑲㈢瓑绲勭箶鍚�30mg妯�(bi膩o)瑷樺悗缃簬闆㈠績绠′腑锛屾寜鐓ц│鍔戠洅閰嶅鐨凞NA鎶芥彁瑭﹀姂鐩掓搷浣滆鏄庢彁鍙朌NA妯℃澘銆�
2.2 瀛樻斁
鐮旂（鍚庣殑妯ｆ湰鎳�(y墨ng)鐩″揩鎻愬彇 DNA锛涘緟娓ǎ鏈噳(y墨ng)閬垮厤鍑嶈瀺銆�
2.3 閬嬭几
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3.1 PCR 妾㈡脯
3.1.1 鎿村瑭﹀姂婧�(zh菙n)鍌欙紙鍦ㄥ弽鎳�(y墨ng)娣峰悎鐗╅厤鍒跺崁(q奴)閫�(j矛n)琛岋級锛�
寰炶│鍔戠洅涓彇鍑虹浉鎳�(y墨ng)鐨� PCR 鍙嶆噳(y墨ng)娑层�乀aq 閰讹紝2000×g 闆㈠績 5 绉掗悩銆傛瘡鍊嬫ǎ鍝佹脯瑭﹀弽鎳�(y墨ng)楂旂郴閰嶅埗瑕嬩笅琛� 2銆�
琛� 2 姣忓�嬫ǎ鍝佹脯瑭﹀弽鎳�(y墨ng)楂旂郴閰嶅埗琛�
3.1.2 鍔犳ǎ锛堟ǎ鏈檿鐞嗗崁(q奴)閫�(j矛n)琛岋級锛�
鍚戞瘡鍊� PCR 绠″瓟涓悇鍒嗚 15μL 鐨勬贩鍚堟恫锛屽啀鍒嗗垾鍔犲叆妯ｆ湰 DNA 妯℃澘 10μL锛岃搵绶婄钃嬶紝500
r/min 闆㈠績 30 s銆�
3.1.3 PCR 妾㈡脯锛堝湪妾㈡脯鍗�(q奴)閫�(j矛n)琛岋級锛� 闅庢锛�95 鈩�/3 min锛�
绗簩闅庢锛�94 鈩�/30 sec锛�60 鈩�/30sec锛�72鈩�/1 min锛�35鍊嬪惊鐠�(hu谩n)锛�
绗笁闅庢锛�72 鈩�/7 min锛� 绗洓闅庢锛�4 鈩� 淇濆瓨
3.1.4 鐡婅剛绯栭浕娉�
鐢ㄩ浕娉崇珐娌栨恫鍒跺倷 1.5%鐨勭搳鑴傜硸鍑濊啝骞虫澘銆傚皣骞虫澘鏀惧叆姘村钩闆绘吵妲斤紝浣块浕娉崇珐娌栨恫鍓涘ソ娌掗亷鑶犻潰锛屽悜 PCR 鎿村鐢�(ch菐n)鐗╀腑鍔犲叆 1/6 楂旂⿳鐨勯浕娉充笂妯ｇ珐娌栨恫锛�6X 涓婃ǎ绶╂矕娑诧級锛屾寜姣斾緥娣峰嫽鍚庡姞鍏ユǎ鍝佸瓟銆傚湪闆绘吵鏅傝ō(sh猫)绔� DNA DL2000 Marker 鍋氬皪鐓с��5 V/cm 闆绘吵绱� 0.5h锛岀暥(d膩ng)婧撮厷钘�(l谩n)鍒伴仈(d谩)涓�瀹氫綅缃檪鍋滄銆傚湪绱鐕堜笅鎴栧嚌鑶犳垚鍍忓剙鐨勭传澶栭�忓皠鍏変笅瑙�瀵熸槸鍚︽摯澧炲嚭闋�(y霉)鏈熺殑鐗圭暟鎬� DNA 闆绘吵甯讹紝鎷嶆敐骞惰閷勩��
4銆佹娓椹�
4.1.1 DNA 鏍搁吀鎻愬彇鎿嶄綔鏂规硶(鍦ㄦǎ鏈檿鐞嗗崁(q奴)閫�(j矛n)琛�)锛�
鍙� n 鍊� 1.5ml 婊呰弻 Eppendorf 绠★紝鍏朵腑 n 鐐哄緟妾㈡ǎ鍝佹暩(sh霉)鍜屼竴绠￠櫚鎬у皪鐓т箣鍜岋紝灏嶆瘡鍊嬬閫�(j矛n)琛岀法铏熸(bi膩o)瑷樸��(娉細瑭﹀姂鐩掍腑鐨勯櫧鎬у皪鐓х洿鎺ヤ綔鐐� PCR 妾㈡脯鐨勬ā鏉匡紝鐒￠渶鎻愬彇鏍搁吀)
4.1.2 姣忕鍔犲叆 100 μl DNA 鎻愬彇娑� 1锛岀劧鍚庡垎鍒ュ姞鍏ュ緟娓ǎ鏈拰闄版�у皪鐓у悇 100μl锛屼竴浠芥ǎ鏈彌鐢ㄤ竴鍊嬪惛闋紱娣峰嫽鍣ㄤ笂闇囪暕娣峰嫽 5 s,浜� 4鈩冿綖25鈩冩浠朵笅锛�12 000 r/min 闆㈠績 10 min銆�
4.1.3 鐩″彲鑳藉惛妫勪笂娓呬笖涓嶇娌夋穩锛屽啀鍔犲叆 10μl DNA 鎻愬彇娑� 2锛屾贩鍕诲櫒涓婇渿钑╂贩鍕� 5s,浜� 4鈩冿綖25鈩� 姊濅欢涓嬶紝2 000 r/min 闆㈠績 10 s銆�
4.1.4 100鈩� 骞叉荡鎴栨哺姘存荡 10 min锛涘姞鍏� 90μl DEPC 姘达紝12 000 r/min 闆㈠績 10 min锛屽惛鍙栦笂娓咃紝 鍗崇偤鎻愬彇鐨� DNA锛屽啺涓婁繚瀛樺緟鐢紙鎻愬彇鐨� DNA 闇�鍦� 2 h 鍏�(n猫i)閫�(j矛n)琛� PCR 鎿村鎴栨斁缃簬-70鈩冨啺绠卞収(n猫i)淇濆瓨锛夈��
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- 01
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