Bac-to-Bac
琛ㄨ揪绯荤粺
I. pFastBac-X
閲嶇粍璐ㄧ矑鐨勬瀯寤?/p>
鍒╃敤鍒嗗瓙鐢熺墿瀛︽妧鏈瀯寤烘墍闇�鐨?/p>
pFastBac
閲嶇粍璐ㄧ矑
,
浠?/p>
DH5伪
涓哄涓?/p>
,
杩涜鎵?/p>
澧?/p>
.(
浠ヤ笅浠?/p>
pFastBac-X
涓轰緥杩涜璇存槑
.
II. pFastBac-x
閲嶇粍璐ㄧ矑鐨勮浆搴ф楠わ細
1.
鍒跺
LB
鐞艰剛骞虫澘銆?/p>
LB
鍩瑰吇娑诧紙鍚?/p>
Agar
锛?/p>
锛氳儼铔嬬櫧鍐?/p>
10g/l
锛?/p>
Nacl 10g/l
锛岄叺姣嶆彁鍙栫墿
5g/l
锛?/p>
Agar 15g/l
楂樺帇鐏弻鍚庯紝鍩瑰吇鍩烘俯搴︿笅闄嶈嚦
60
鈩冨乏鍙宠繀閫熷姞鍏ヤ笅鍒楁垚鍒嗭細
鍗￠偅闇夌礌
50ug/ml
搴嗗ぇ闇夌礌
7ug/ml
鍥涚幆绱?/p>
10ug/ml
Bluo-gal 100ug/ml
IPTG 40ug/ml
鍚勬垚鍒嗘贩鍖�鍚庯紝涔樼儹鍦ㄦ瘡鍧楀钩鏉夸腑鍔犲叆绾?/p>
20ml
鍩瑰吇鍩猴紝寰呭嚌鍥哄悗鏀剧疆
37
鈩冪儤绠?/p>
骞茬嚗浜х敓鐨勬按姹姐�?/p>
2.
鍙栧嚭
DH10BacTM
鎰熷彈鎬佺粏鑳炲啺涓婅瀺瑙ｃ�?/p>
3.
鐢ㄧЩ娑叉灙鍚稿彇
100ulDH10BacTM
鎰熷彈鎬佺粏鑳炰簬
1.5ml EP
绠°�?/p>
4.
杞昏交鍔犲叆
1ng
锛堢害
5ul
锛?/p>
閲嶇粍璐ㄧ矑
X
浜庢劅鍙楁�佺粏鑳炰腑锛?/p>
杞昏交鏁叉墦绠″浣夸箣娣峰寑銆?/p>
5.
鍐颁笂鏀剧疆
30min
銆?/p>
6. 42
鈩冨惊鐜按娴撮潤姝?/p>
45
绉掋�?/p>
7.
蹇�熸斁缃啺涓?/p>
2min
銆?/p>
8.
鍦ㄤ笂杩扮涓姞鍏?/p>
900ul S.O.C.
鍩瑰吇鍩恒�?/p>
9.
灏?/p>
EP
绠＄疆浜?/p>
37
鈩冩憞搴婏紙
225rpm
锛?/p>
4h
銆?/p>
10.
鐢?/p>
S.O.C.
绋�閲婁笂杩扮粏鑳炶嚦
10
锛?/p>
1
銆?/p>
10
锛?/p>
2
銆?/p>
10
锛?/p>
3
銆?/p>
11.
鍦ㄦ瘡鍧?/p>
LB
鍩瑰吇鍩轰腑鍔犲叆涓婅堪绋�閲婃恫
100ul
锛屾秱甯冨潎鍖�銆?/p>
12. 37
鈩冨煿鍏?/p>
24
锛?/p>
48h
銆?/p>
锛堝崟鍏嬮殕寰堝皬锛?/p>
24h
鍓嶅緢闅惧垎杈ㄦ槸鍚︿负钃濊壊鍏嬮殕锛?/p>
娉細
1
锛?/p>
1
銆?/p>
3
銆?/p>
4
銆?/p>
8
銆?/p>
10
銆?/p>
11
鍦ㄨ秴鍑�鍙板唴杩涜銆?/p>
2
锛?/p>
鐢?/p>
X
锛?/p>
gal
浠ｆ浛
Bluo-gal
浼氶檷浣庨鑹叉祿搴︺�?/p>
鐪熸鐨勭櫧鑹插厠闅嗗湪鑿岃惤闀垮緱杈冨ぇ鏃?/p>
杩樻槸鍛堢櫧鑹诧紝寤鸿鍦ㄩ粦鑹茶儗鏅笅杩涜鎸戦�夈�?/p>
III.
閲嶇粍
Bacmid DNA
鐨勫垎绂绘彁鍙?/p>
锛堝湪鎻愬彇鍓嶏紝鍙互鍦ㄥ惈
Bluo-gal
鐨?/p>
LB
鐞艰剛骞虫澘涓婂垝鏉胯繘涓�姝ョ‘璁わ級
婧舵恫閰嶇疆锛氭憾娑测厾锛?/p>
Tris-Hcl(15Mm) 0.119g
EDTA (10Mm) 0.187g
Rnase (100ug/ml) 0.5ml(
璐娑?/p>
10mg/ml)
鐢ㄧ函姘存憾瑙ｅ苟瀹氬鑷?/p>
50ml
锛堣皟
pH8.0
锛?/p>
婧舵恫鈪★細
NaOH 0.2N
SDS 1%
婧舵恫鈪細閱嬮吀閽撅紙
3M
锛?/p>
14.7g
婧惰В瀹氬鑷?/p>
50ml
锛?/p>
pH5.5
TE
婧舵恫锛?/p>
1.
鎸戝彇鐧借壊鍏嬮殕浜?/p>
2mlLB
鍩瑰吇鍩猴紙鍚?/p>
50ug/ml
鍗￠偅闇夌礌銆?/p>
7ug/ml
搴嗗ぇ闇夌礌銆?/p>
10ug/ml
鍥涚幆绱狅級
锛?/p>
37
鈩冩憞搴?/p>
250
锛?/p>
300rpm
鍩瑰吇
24h
浠ヤ笂銆?/p>