涓€銆?/span>瀹為獙璇曞墏锛欬/span>

1.CTAB鎶芥彁娑诧細

2%锛圏/span>w/v锛堻/span>CTAB

100 mmol/L Tris-HCl(pH8.0)

20 mmol/L EDTA(pH8.0)

1.4 mol/L NaCl

鎶芥彁鍚閰氳緝澶氱殑妞嶇墿鏉愭枡鏃讹紙姣斿鏈ㄦ湰妞嶇墿锛夛紝涓婅堪鎶芥彁娑蹭腑鍙彟鍔犲叆2%鐨凕/span>PVP锛堣仛涔欑儻鍚″挴鐑烽叜锛夈€侟/span>

鎶芥彁娑蹭簬瀹ゆ俯淇濆瓨锛屽彲鍦ㄥ嚑骞村唴淇濇寔绋冲畾銆備复鐢ㄤ箣鍓嶅悜瑁呮湁涓婅堪鎶芥彁娑茬殑璇曠涓姞鍏ョ害2%-3%锛圏/span>v/v锛夌殑尾-宸熀涔欓唶銆侟/span>

2銆侀唻閰搁挔锛欬/span>

3 mol/L NaAc

鐢ㄥ啺涔欓吀璋傸/span>pH鍊艰嚦4.8-5.2銆侟/span>

3銆?/span>TE婧舵恫锛欬/span>

4銆佸叾瀹冿細

鏃犳按涔欓唶锛屽紓涓欓唶锛孅/span>75锛呬箼閱囷紝閰氾細姘豢锛氬紓鎴婇唶锛圏/span>25锛欬/span>24锛欬/span>1锛夛紝姘豢锛氬紓鎴婇唶锛圏/span>24锛欬/span>1锛夛紝尾-宸熀涔欓唶锛岄噸钂告按銆侟/span>

浜屻€?/span>瀹為獙姝ラ

1.鏍峰搧鐮旂（

1)鍙朁/span>1g鐨勬牱鍝佹斁鍏?/span>2ml ep绠′腑

2)鍔犲叆1-2绮扅/span>5mm鐮旂（鐝燑/span>

3)鍔犲叆1ml鐨凕/span>CTAB鎶芥彁娑秉/span>

4)灏嗗姞鏈夋牱鍝佺殑ep绠℃斁鍏ラ珮閫氶噺缁勭粐鐮旂（浠殑閫傞厤鍣ㄤ腑锛岀洊濂键/span>

5)灏嗙爺纾ㄨ浆閫熻皟鍒?/span>1800锛岃瀹氱爺纾ㄦ椂闂翠负90绉掞紙鍙牴妞愭牱鍝佺殑鐮旂（鐨勯毦鏄撶▼搴︽潵璋冭妭锛屾鏁版嵁鏄互閾舵潖钀藉彾涓烘牱鏈緱鍑猴級

6)鍚姩鐮旂（浠鏍峰搧杩涜鐮旂（锛堟牴鎹笉鍚岀殑鏍峰搧锛屾湰韬€ц川涓嶅悓锛岄渶瑕佸鐮旂（棰戠巼鍜岀爺纾ㄦ椂闂磋繘琛岄€傚綋鐨勮皟鏁达級

2. DNA鐨勭矖鎻忺/span>

1)灏嗙爺纾ㄥソ鐨勬牱鍝佸彇鍑轰簬65鈩傸/span>姘存荡45min锛屼腑閫旈棿闅旓紙杞绘煍锛夋尟鑽′笁娆℃贩鍖€

2)鍐峰嵈鍚庡姞鍏ョ瓑浣撶Н姘豢锛氬紓鎴婇唶锛圏/span>24锛欬/span>1锛夛紝杞绘煍棰犲€掓贩鍖€浣夸钩鍖朁/span>10min

3) 7000g-8000g绂诲績10min锛圏/span>18-20鈩傸/span>锛堻/span>

4)鍚稿彇涓婃竻浜庡彟涓€骞插噣鐨勭蹇冪涓?/span>

5)锛堟牴鎹渶瑕佸彲鐢ㄦ隘浠匡細寮傛垔閱囧涓婃硶閲嶅鎶芥彁涓€娆★紝鍚稿彇涓婃竻锛堻/span>

6)鍔犲叆涓庝笂娓呮恫绛変綋绉紙涓斿凡浜嶞/span>-20鈩傸/span>棰勫喎鐨勫紓涓欓唶锛岄鍊掓贩鍖€锛岀害1鑷颤/span>2鍒嗛挓鍚庡簲鏈夌櫧鑹茬诞鐘舵矇娣€鍑虹幇锛堣嫢娌℃湁锛屽垯鍔犲叆涓婃竻娑秉/span>1/5鑷颤/span>1/10浣撶Н鐨凕/span>pH4.8-5.2鐨凕/span>3M NaAc,娣峰寑锛屼簬锛岦/span>20鈩傸/span>鍐扮涓矇娣€30min锛堻/span>

7)绂诲績娉曟矇娣€DNA

8)鍦?/span>75%涔欓唶涓紓娲桙/span>DNA鏁板垎閽熶互涓婏紝鐢?/span>Tip澶村惛骞蹭箼閱嘃/span>

9)鐢?/span>1ml 75%涔欓唶鍐嶆紓娲椾竴娆狘/span>

10)娌夋穩浜庡娓╂垨64鈩傸/span>浠ヤ笅鐨勬亽娓╃涓共鐕ョ墖鍒昏嚦鍒氬嚭鐜板崐閫忔槑锛屼笉瑕佽繃搴﹀共鐕?/span>

11)鐢?/span>50渭lTE婧惰В娌夋穩锛屽彲鐢?/span>65鈩傸/span>姘存荡鍔╂憾锛孅/span>DNA绮楁彁鐗╁彲鐢ㄤ簬绮楃暐PCR绛夈€侟/span>

3. DNA鐨勭函鍖朁/span>

1)鍚慄/span>TE婧惰В鐨凕/span>DNA绮楁彁鐗╀腑鍏?/span>4-10渭l RNaseA(绾?/span>40-100渭g)

2)浜嶞/span>37鈩傸/span>閰惰ВRNA 1hr鎴朁/span>65鈩傸/span>閰惰ВRNA 30min浠ヤ笂

3)鍔犲叆50渭l閰氾細姘豢锛氬紓鎴婇唶锛圏/span>25锛欬/span>24锛欬/span>1锛夛紝杞昏交棰犲€掓贩鍖€10min

4) 10000g浠ヤ笂甯告俯绂诲績10min锛屽惛鍙栦笂娓匋/span>

5)鍔犲叆50渭l浠挎隘锛氬紓鎴婇唶锛圏/span>24锛欬/span>1锛夎交杞婚鍊扅/span>10min

6) 10000g浠ヤ笂甯告俯绂诲績10min锛屽惛鍙栦笂娓匋/span>(鏍规嵁闇€瑕佸彲閲嶅鐢ㄦ隘浠匡細寮傛垔閱囧涓婃硶鍐嶆娊鎻忺/span>1-2娆★紝浠ュ厖鍒嗗幓闄よ泲鐧藉拰閰欬/span>)

7)鍚戜笂娓呬腑鍔犲叆1/5-1/10浣撶Н鐨凕/span>pH4.8-5.2鐨凕/span>3M NaAc,骞跺姞鍏?/span>2.5鍊嶄綋绉棤姘翠箼閱囷紝浜庯紞20鈩傸/span>娌夋穩30min浠ヤ笂

8) 12000g銆?/span>4鈩傸/span>浣庢俯绂诲績10min,鍚稿共娑蹭綋

9)娌夋穩鐢?/span>75锛呬箼閱囨紓娲椾簩娆狘/span>

10)娌夋穩浜庡娓╂垨64鈩傸/span>浠ヤ笅鎭掓俯绠变腑骞茬嚗鐗囧埢鑷冲垰寮€濮嬪嚭鐜板崐閫忔槑鐘讹紝鍕胯繃搴﹀共鐕ュ姞鍏?/span>50渭l閲嶈捀姘存憾瑙Ⅻ/span>DNA锛屽彲浜嶞/span>65鈩傸/span>姘存荡鍔╂憾

11)鍙朁/span>5渭l DNA鐢垫吵妫€鏌?/span>DNA鐨勮川閲廃/span>

12) 100-500鍊嶇█閲婂悗浜庡垎鍏夊厜搴﹁涓婃祴瀹欬/span>OD260鍙夵/span>OD280锛屽垎鏋忺/span>DNA鐨勬祿搴﹀拰璐ㄩ噺

13)淇濆瓨DNA浜嶞/span>-20鈩傸/span>

琛ュ厖锛氬鏋滈渶瑕佹彁鍙栫殑鏍峰搧姣旇緝澶氾紙姣斿1g锛夛紝鐮旂（濂界殑鏍峰搧鍦ㄥ悗缁殑鎻愬彇涓紝闇€瑕佽浆绉诲埌澶х殑epp绠′腑锛岀爺纾ㄦ椂鍙互鍔犲叆杈冨皯鐨勬娊鎻愭恫锛岀爺纾ㄥ畬姣曟寜鐓?/span>1g鏍峰搧10ml鎻愬彇娑茶ˉ鍏呮娊鎻愭恫锛岃繘琛屽悗缁殑鎿嶄綔銆侟/span>