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308047410新蟲 (小有名氣)
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[求助]
CODEHOP 結(jié)果怎么看以及簡(jiǎn)并引物設(shè)計(jì)相關(guān)問題 已有2人參與
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CODEHOP 結(jié)果怎么看？ 下面是我用CODEHOP設(shè)計(jì)簡(jiǎn)并引物的結(jié)果，1、不知道怎么選擇，A、B、C、D、中哪個(gè)是上游引物，哪個(gè)是下游引物？怎么知道簡(jiǎn)并引物之間的距離？引物的選擇有什么要求？ 另外2、在BLOCKMAKE 時(shí)輸入的蛋白序列是全長(zhǎng)?還是找到保守區(qū)域后的序列？ 3、在CODEHOP階段，沒有本物種的密碼子表，選擇跟本物種不同目的物種可以嗎？ 4、篩選出引物之后還需要做哪些工作，需不需要驗(yàn)證引物？怎么驗(yàn)證？ 求各位大師知道，感激不盡！ CODEHOP Results Oligo Summary Not all overlapping primers are shown Warning: All weights in the block were less than or equal to zero. Warning: Computing position-based sequence weights. CODEHOP Version 10/14/04.1 COPYRIGHT 1997-2004, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Parameters: Amino acids PSSM calculated with odds ratios normalized to 100 and back-translated with Standard genetic code and codon usage table "../docs/shrimp.codon.use" Maximum core degeneracy 128 Core strictness 0.00 Clamp strictness 1.00 Target clamp temperature 60.00 C DNA Concentration 50.00 nM Salt Concentration 50.00 mM Codon boundary 0 Most common codon 0 Verbose 0 Output 3 Begin 1 PolyX 5 Suggested CODEHOPS: The degenerate region (core) is printed in lower case, the non-degenerate region (clamp) is printed in upper case. Block unknown__A Oligos W G R P E D M T M D R P S W K I D S E A P G T D L A A E T A A A L A A A S I V F Q D GACCTGGCCGCCgaracngcngc -3' Core: degen=32 len=11 Clamp: score=72, len=12 temp= 61.6 GCACCGACCTGGCCgsngaracngc -3' Core: degen=64 len=11 Clamp: score=76, len=14 temp= 63.7 CGGCACCGACCTGgcngsngarac -3' Core: degen=64 len=11 Clamp: score=74, len=13 temp= 63.7 TGGACCGGCCCTGCkrbaarathga -3' Core: degen=72 len=11 Clamp: score=65, len=14 temp= 61.5 GGGCCGGCCCgarganatgac -3' Core: degen=8 len=11 Clamp: score=71, len=10 temp= 62.6 TGGGGCCGGCCngarganatga -3' Core: degen=32 len=11 Clamp: score=80, len=11 temp= 57.5 *** CLAMP NEEDS EXTENSION TGGGGCCGGccngarganatg -3' Core: degen=32 len=12 Clamp: score=70, len=9 temp= 57.5 *** CLAMP NEEDS EXTENSION TGGGGCCGGccngarganat -3' Core: degen=32 len=11 Clamp: score=70, len=9 temp= 57.5 *** CLAMP NEEDS EXTENSION Complement of Block unknown__A Oligos W G R P E D M T M D R P S W K I D S E A P G T D L A A E T A A A L A A A S I V F Q D ggnctyctntaCTGGTACCTGGCCGG -5' Core: degen=32 len=11 Clamp: score=78, len=15 temp= 62.8 gnctyctntactGGTACCTGGCCGG -5' Core: degen=32 len=12 Clamp: score=71, len=13 temp= 62.8 ctyctntactgGTACCTGGCCGG -5' Core: degen=8 len=11 Clamp: score=66, len=12 temp= 62.8 ttytadctrhbGCTCCTGGGGCCGT -5' Core: degen=108 len=11 Clamp: score=57, len=14 temp= 61.5 cgncsnctytgGCGGCGGCGG -5' Core: degen=64 len=11 Clamp: score=75, len=10 temp= 62.9 csnctytgncgGCGGCGGGAC -5' Core: degen=64 len=11 Clamp: score=73, len=10 temp= 60.6 ctytgncgncgGCGGGACCGGC -5' Core: degen=32 len=11 Clamp: score=71, len=11 temp= 65.1 Block unknown__B Oligos W Q D E L P W G A C W L Y R A T G E E S Y M T Q A Q G Y I E E F G F GCTGGCTGTACCGGgcnacnggnga -3' Core: degen=64 len=11 Clamp: score=72, len=14 temp= 61.4 GGCCTGGGGCGCCykntggytnta -3' Core: degen=128 len=11 Clamp: score=74, len=13 temp= 63.7 TGGCCTGGGGCgcnykntggyt -3' Core: degen=128 len=11 Clamp: score=71, len=11 temp= 62.8 TGGsangaygaryt -3' Core: degen=64 len=11 Clamp: score=66, len=3 temp=-100.4 *** CLAMP NEEDS EXTENSION Complement of Block unknown__B Oligos W Q D E L P W G A C W L Y R A T G E E S Y M T Q A Q G Y I E E F G F acccsncgnrmGACCGACATGGCCCGG -5' Core: degen=128 len=11 Clamp: score=82, len=16 temp= 61.3 cgnrmnaccraCATGGCCCGGTGGC -5' Core: degen=128 len=11 Clamp: score=73, len=14 temp= 61.1 cgntgnccnctCCTCCGGATGAACTGGCT -5' Core: degen=64 len=11 Clamp: score=61, len=18 temp= 61.7 Block unknown__C Oligos N F D W D D K T A G C Y A L M A E L D AACTTCGACtgggaygwnaa -3' Core: degen=16 len=11 Clamp: score=77, len=9 temp= 43.3 *** CLAMP NEEDS EXTENSION AACTTCGAytgggaygwna -3' Core: degen=32 len=11 Clamp: score=80, len=8 temp= 1.2 *** CLAMP NEEDS EXTENSION AACTTnkaytgggayg -3' Core: degen=32 len=11 Clamp: score=82, len=5 temp=-50.5 *** CLAMP NEEDS EXTENSION AACktnkaytggga -3' Core: degen=32 len=11 Clamp: score=77, len=3 temp=-118.0 *** CLAMP NEEDS EXTENSION Complement of Block unknown__C Oligos N F D W D D K T A G C Y A L M A E L D anmtraccctrcTGTTCTGGCGGCCGA -5' Core: degen=32 len=12 Clamp: score=75, len=15 temp= 61.0 traccctrcwntTCTGGCGGCCGACG -5' Core: degen=32 len=12 Clamp: score=72, len=14 temp= 62.2 accctrcwnttCTGGCGGCCGACG -5' Core: degen=16 len=11 Clamp: score=68, len=13 temp= 62.2 Block unknown__D Oligos T P L G M I Y I M D W G S L R H A M N V A F I A A R A A S L G I D T E E Y N GCACGCCATGAACGTGgsnttyrtngc -3' Core: degen=128 len=11 Clamp: score=69, len=16 temp= 63.5 Complement of Block unknown__D Oligos T P L G M I Y I M D W G S L R H A M N V A F I A A R A A S L G I D T E E Y N csnaaryancgGCAGGCCCGGC -5' Core: degen=128 len=11 Clamp: score=56, len=11 temp= 64.9 Block unknown__E Oligos N W A Q T Q I D Y A L G S T G H S Y M C G F G E N P P TGTGCGGCTTCGGCrmnaayccncc -3' Core: degen=128 len=11 Clamp: score=80, len=14 temp= 60.2 ACATGTGCGGCTTCggnrmnaaycc -3' Core: degen=128 len=11 Clamp: score=80, len=14 temp= 60.4 CACTCCTACATGTGCGGCttyggnrmnaa -3' Core: degen=128 len=11 Clamp: score=74, len=18 temp= 61.3 CCATGGGCCACTCCTACATGkknggnttygg -3' Core: degen=128 len=11 Clamp: score=73, len=20 temp= 63.6 GCCCAGACCCAGATCgaytayrcnbt -3' Core: degen=96 len=11 Clamp: score=76, len=15 temp= 60.8 TGGGCCCAGACCCAGryngaytayrc -3' Core: degen=128 len=11 Clamp: score=78, len=15 temp= 60.1 CTGGGCCCAGACCcarryngayta -3' Core: degen=64 len=11 Clamp: score=73, len=13 temp= 60.1 Complement of Block unknown__E Oligos N W A Q T Q I D Y A L G S T G H S Y M C G F G E N P P gtyyrnctratGCGGGACCCGAGGT -5' Core: degen=64 len=11 Clamp: score=69, len=14 temp= 62.5 ctratrygnvaCCCGAGGTACCCGG -5' Core: degen=96 len=11 Clamp: score=66, len=14 temp= 62.8 ccnaarccnykCTTGGGTGGT -5' Core: degen=128 len=11 Clamp: score=78, len=10 temp= 27.1 *** CLAMP NEEDS EXTENSION aarccnyknttGGGTGGT -5' Core: degen=128 len=11 Clamp: score=80, len=7 temp= 21.1 *** CLAMP NEEDS EXTENSION ccnyknttrggTGGT -5' Core: degen=128 len=11 Clamp: score=66, len=4 temp=-14.8 *** CLAMP NEEDS EXTENSION Block unknown__ X M K A W I D M P M L V P F D F Y D Q E Y P G C D G C G M T P Y D Y Y Q Y S No suggested primers found. Complement of Block unknown__ X M K A W I D M P M L V P F D F Y D Q E Y P G C D G C G M T P Y D Y Y Q Y S No suggested primers found. Oligos Degenerate alphabet Block unknown__A W G R P E D M oligo:5'-TGGGGCCGGccngarganat-3' degen=32 temp=57.5 Extend clamp W G R P E D M oligo:5'-TGGGGCCGGccngarganatg-3' degen=32 temp=57.5 Extend clamp W G R P E D M T oligo:5'-TGGGGCCGGCCngarganatga-3' degen=32 temp=57.5 Extend clamp G R P E D M T oligo:5'-GGGCCGGCCCgarganatgac-3' degen=8 temp=62.6 D R P S W K I D oligo:5'-TGGACCGGCCCTGCkrbaarathga-3' degen=72 temp=61.5 G T D L A A E T oligo:5'-CGGCACCGACCTGgcngsngarac-3' degen=64 temp=63.7 T D L A A E T A oligo:5'-GCACCGACCTGGCCgsngaracngc-3' degen=64 temp=63.7 D L A A E T A A oligo:5'-GACCTGGCCGCCgaracngcngc-3' degen=32 temp=61.6 Complement of Block unknown__A P E D M T M D R P ggnctyctntaCTGGTACCTGGCCGG oligo:5'-GGCCGGTCCATGGTCatntcytcngg-3' degen=32 temp=62.8 E D M T M D R P gnctyctntactGGTACCTGGCCGG oligo:5'-GGCCGGTCCATGGtcatntcytcng-3' degen=32 temp=62.8 E D M T M D R P ctyctntactgGTACCTGGCCGG oligo:5'-GGCCGGTCCATGgtcatntcytc-3' degen=8 temp=62.8 K I D S E A P G T ttytadctrhbGCTCCTGGGGCCGT oligo:5'-TGCCGGGGTCCTCGbhrtcdatytt-3' degen=108 temp=61.5 A A E T A A A cgncsnctytgGCGGCGGCGG oligo:5'-GGCGGCGGCGgtytcnscngc-3' degen=64 temp=62.9 A E T A A A L csnctytgncgGCGGCGGGAC oligo:5'-CAGGGCGGCGgcngtytcnsc-3' degen=64 temp=60.6 E T A A A L A A ctytgncgncgGCGGGACCGGC oligo:5'-CGGCCAGGGCGgcngcngtytc-3' degen=32 temp=65.1 Block unknown__B W Q D E L oligo:5'-TGGsangaygaryt-3' degen=64 temp=-100.4 Extend clamp P W G A C W L oligo:5'-TGGCCTGGGGCgcnykntggyt-3' degen=128 temp=62.8 P W G A C W L Y oligo:5'-GGCCTGGGGCGCCykntggytnta-3' degen=128 temp=63.7 W L Y R A T G E oligo:5'-GCTGGCTGTACCGGgcnacnggnga-3' degen=64 temp=61.4 Complement of Block unknown__B W G A C W L Y R A acccsncgnrmGACCGACATGGCCCGG oligo:5'-GGCCCGGTACAGCCAGmrngcnsccca-3' degen=128 temp=61.3 A C W L Y R A T G cgnrmnaccraCATGGCCCGGTGGC oligo:5'-CGGTGGCCCGGTACarccanmrngc-3' degen=128 temp=61.1 A T G E E S Y M T Q cgntgnccnctCCTCCGGATGAACTGGCT oligo:5'-TCGGTCAAGTAGGCCTCCtcnccngtngc-3' degen=64 temp=61.7 Block unknown__C N F D W D oligo:5'-AACktnkaytggga-3' degen=32 temp=-118.0 Extend clamp N F D W D D oligo:5'-AACTTnkaytgggayg-3' degen=32 temp=-50.5 Extend clamp N F D W D D K oligo:5'-AACTTCGAytgggaygwna-3' degen=32 temp=1.2 Extend clamp N F D W D D K oligo:5'-AACTTCGACtgggaygwnaa-3' degen=16 temp=43.3 Extend clamp Complement of Block unknown__C D W D D K T A G C anmtraccctrcTGTTCTGGCGGCCGA oligo:5'-AGCCGGCGGTCTTGTcrtcccartmna-3' degen=32 temp=61.0 W D D K T A G C traccctrcwntTCTGGCGGCCGACG oligo:5'-GCAGCCGGCGGTCTtnwcrtcccart-3' degen=32 temp=62.2 W D D K T A G C accctrcwnttCTGGCGGCCGACG oligo:5'-GCAGCCGGCGGTCttnwcrtccca-3' degen=16 temp=62.2 Block unknown__D H A M N V A F I A oligo:5'-GCACGCCATGAACGTGgsnttyrtngc-3' degen=128 temp=63.5 Complement of Block unknown__D A F I A A R A A csnaaryancgGCAGGCCCGGC oligo:5'-CGGCCCGGACGgcnayraansc-3' degen=128 temp=64.9 Block unknown__E W A Q T Q I D Y oligo:5'-CTGGGCCCAGACCcarryngayta-3' degen=64 temp=60.1 W A Q T Q I D Y A oligo:5'-TGGGCCCAGACCCAGryngaytayrc-3' degen=128 temp=60.1 A Q T Q I D Y A L oligo:5'-GCCCAGACCCAGATCgaytayrcnbt-3' degen=96 temp=60.8 T G H S Y M C G F G oligo:5'-CCATGGGCCACTCCTACATGkknggnttygg-3' degen=128 temp=63.6 H S Y M C G F G E N oligo:5'-CACTCCTACATGTGCGGCttyggnrmnaa-3' degen=128 temp=61.3 M C G F G E N P oligo:5'-ACATGTGCGGCTTCggnrmnaaycc-3' degen=128 temp=60.4 C G F G E N P P oligo:5'-TGTGCGGCTTCGGCrmnaayccncc-3' degen=128 temp=60.2 Complement of Block unknown__E Q I D Y A L G S T gtyyrnctratGCGGGACCCGAGGT oligo:5'-TGGAGCCCAGGGCGtartcnryytg-3' degen=64 temp=62.5 D Y A L G S T G H ctratrygnvaCCCGAGGTACCCGG oligo:5'-GGCCCATGGAGCCCavngyrtartc-3' degen=96 temp=62.8 G F G E N P P ccnaarccnykCTTGGGTGGT oligo:5'-TGGTGGGTTCkynccraancc-3' degen=128 temp=27.1 Extend clamp F G E N P P aarccnyknttGGGTGGT oligo:5'-TGGTGGGttnkynccraa-3' degen=128 temp=21.1 Extend clamp G E N P P ccnyknttrggTGGT oligo:5'-TGGTggrttnkyncc-3' degen=128 temp=-14.8 Extend clamp |
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308047410
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7樓2015-05-21 21:48:02
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308047410: 金幣+5 2015-05-21 21:48:19
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308047410: 金幣+5 2015-05-21 21:48:19
| 1，先根據(jù)你比對(duì)的氨基酸位置確定引物上下游，根據(jù)一個(gè)氨基酸密碼子對(duì)應(yīng)3個(gè)堿基的關(guān)系，就可以確定上下游引物的距離。2，設(shè)計(jì)好的引物可以再oligo里評(píng)價(jià)一下，3，如果是幾個(gè)蛋白質(zhì)比對(duì)之后設(shè)引物的話，應(yīng)該就是找保守區(qū)設(shè)引物，如果只是單純找一個(gè)蛋白，想擴(kuò)增背后的基因，可以找它的cds設(shè)計(jì)引物，4，引物驗(yàn)證的話，可以做PCR，然后產(chǎn)物割膠測(cè)序，看看。5，我也是新手，最近一直摸索，這是我的一些想法，有不對(duì)的地方請(qǐng)?jiān)彙！? |
6樓2015-05-21 17:54:00
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