请教启动子CAG的扩增，引物与具体参数怎么设定？？

leozarah

本人最近想扩增CAG启动子，但试了很多次都没能成功，包括设定长引物，用高GCbuffer酶，加DMSO,都没能P出来，所以希望有高手能够指点一下，附下CAG的序列：GTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATAT& n4 N  e, X; _6 ~% y8 h4 e9 Z
GGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTG
% l6 D, b' J8 _: L. zACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACT
' k, M# y+ y0 d7 R6 i7 Q5 [ATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGT$ S, J/ T0 |" ~0 Y3 y1 a. p
CAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAG
# Z6 T& K! T2 m. r$ q! l! g2 |TACATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCC) w# |/ D2 G1 f% V' D# y
ATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGG
' t& L8 o# s& O) ]% Q6 HCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAG/ G# ?) U5 n1 V1 V  l
GTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCG
+ b1 k9 Y( Z6 x$ d' QGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGC) O3 s' K( i) L/ ?9 [' n0 {
GCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCC
; b: z) I2 F( QCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAG# e( t" {6 v* M; q
CCTTAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTG
$ o$ x/ l, O4 yCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTT
6 ]; ^. A* V% t" _* {6 K; `GTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGG
" y7 l# w0 R3 n& m  `1 Y; M* ZGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCT3 V4 H8 {4 e1 Q, l, W) S% Y. e
GTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTGC
8 `1 t1 r1 q. r8 H- L4 V" LGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGG7 W/ {! P3 A6 s0 {* w
CCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGC3 [& c' B! m/ p; r) ?8 g9 E
GGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATC# \. F/ t1 Q3 T9 J
TGGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCGAAGCGGTGCGGCGCC
/ s- Y2 R# v; d1 yGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCATCTCCAGC
8 m6 X# I$ I' C- U+ y/ KCTCGGGGCTGCCGCAGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGT
# w. n/ Y7 D1 jGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCA# }# @# U' W1 n- d& s" Z
ACGTGCTGGTTGTTGTGCTGTCTCATCATTTTGGCAAAGAATTCCTCGAGGATATCgcggccgctacgta
1 m( A& t' T2 o1 o5 uaattccgccccccccccccctctccctcccccccccctaacgttactggccgaagccgcttggaataagg* r6 s0 T8 w$ L, A- A
ccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaa% Z5 a) j2 y9 b
cctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgt
+ x9 K  W5 n/ U- T* vtgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttg4 X9 c  P0 C& X( N  m- W. W
caggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacct  `6 b' K% Z- |: F4 Z
gcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcct( u9 A  d( W; ?0 }9 @4 J; j$ w
caagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcc' p5 W$ {- ]+ w# a/ O0 Y+ ~( F
tcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggac  Y* J8 {$ J! A% z) ~
gtggttttcctttgaaaaacacgatgataatatggccacaaccatggtgagcaagggcgaggagctgttc8 t8 |4 d5 |$ X! d) M
accggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcg
$ i: n# K/ j# r5 c' Kagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgt& W6 W0 H' ]& `, q# t
gccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatg4 V3 q" v% c: {! s5 N$ L  W: ?+ z
aagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaagg
! ^$ q8 c8 H7 V2 Q# bacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagct
" j- \$ b5 N  S/ m8 j6 z& _gaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccac
0 _$ l7 n, _7 _% F$ f* C1 Raacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcg) r# r/ b0 [- ~
aggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgct
9 m+ Q0 F% G; R1 h+ G2 h" Q. ggcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatg
1 v4 e2 ]# n9 ^2 a8 P! Q6 |$ `gtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaagcggcc
* ], B* d) g, t1 TggccgccagcacagtggtcgatcgacctgcaggcatgcaagcttcagCCAATGCCCTGGCTCACAAATAC# I0 ~" w- [. P1 F
CACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTG
/ r6 P/ v5 X2 j, H8 VGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGAC
$ H( t0 `" v- \7 k9 [+ {! k4 EATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCATA
6 ?9 Q+ A3 m8 H( i% Q4 F0 GTGCTGGCTGCCATGAACAAAGGTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCAT+ z, H4 N: o# z  Q( b* l
TCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTT: M0 X6 Z  J9 Z2 K) R
CTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCC$ a, P  Y7 ^7 g/ d, S' o2 h
AGTCATAGCTGTCCCTCTTCTCTTATGAAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGGTC4 B) ~% N: @0 y+ C% y
ATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAG
3 y/ ?0 ~9 M( hTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCC  b# i- I) W( J: I, v& i$ ^- j; H
AGTCGGGAAACCTGTCGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACT: t  x6 J+ l* s1 l" S
CCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTA
  B/ n2 }  B, Z; [  Q' jTTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGG3 G6 g# o( @, d
CCTAGGCTTTTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCA
' h4 x6 E2 T4 O) PCAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATC
! m$ q0 b! x( D% l6 \4 F% R* uTTATCATGTCTGGATCCGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGC3 a" l% {' C# ~3 k* I8 H
GCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCA
1 Y: A8 x/ L8 L/ W$ a4 n1 c/ A: lCTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGC7 {( F) a0 ?. |8 }, r. g
CAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACG! }- p  \% v4 R% Q% o
AGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTT' z7 D4 I+ w. }
TCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTT9 @3 _. Y/ K) q. ~: J, D# n$ _- N/ _
CTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTC. r8 r- `( @* d
GCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCG
, r3 M' `8 Y  K) O. u$ v" M. k5 xTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGA
7 X) [0 E% w. g0 {* E* E1 ^9 WGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAG( h+ ?( K& h+ t" `. O4 |2 j  b
TATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAA/ f. ~: A/ F9 `/ j
ACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCT. G2 [& _! j: ^$ p! q
CAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTT& \; [  ^3 r* |+ N: A; ^( }
TGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAAT2 y, W, j9 k, M1 q; B9 j5 @
CTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCG0 W+ P2 j8 K' @1 S3 r
ATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCT# L, _$ y( P# N$ e( [
TACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAAT- E, A. C) j& w
AAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATT
+ l5 R! P# V3 T) MAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTA. {# l9 v. n, b% h
CAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCG
, D7 {  w& M, E8 w- }- P( n2 k/ \AGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGT
# U2 M+ ?( |  n: C9 l& K; ]0 N3 FAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCG
2 S9 D5 Z0 \2 H1 z& Q. R3 aTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAG
" p  l1 f$ Q4 p- Q2 }5 \& j" z# @TTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATT* I6 f& j. I2 b& |( I$ g& ]
GGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCA
) t! n) n; f- d; F4 bCTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAG
; H  r4 |4 s$ iGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAA
% O! Y) f% b: o4 v6 C! lTATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATA" S, g0 I* }' k- h. K- J+ d9 ^+ C
AACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTG
/ a9 ?1 T0 X$ U  ?% B3 ^9 U; o

woaiIPS

CAG启动子GC含量很高  PCR很难 即使用NEB GCbuffer优化酶也难 所以建议最好从骨架再提 酶切连接

leozarah

回复 woaiIPS 的帖子: T& C" j; N/ o" t# R3 U

. [7 w! r4 {6 `! V' n嗯，GC含量确实很高，但我们手上好几个模版都没有太好的酶切位点。用了好几种酶，试过梯度退火和加DMSO/甲酰胺，但最理想的情况也只能p出1200bp左右的条带（目的片段1800bp左右），估计是跳过了几段高GC含量的区域，上次有看到帖友讨论过，所以想请教下论坛里面的高手~~