紧急请教！！请高手帮我看看这两个引物(引物|引物二聚体) - PCR实验 - 生物秀
标题: 紧急请教！！请高手帮我看看这两个引物(引物|引物二聚体)
摘要: [紧急请教！！请高手帮我看看这两个引物(引物|引物二聚体)] 请高手帮我看看这两个引物，用PRIMER PREMIER搜索的，OLIGO验证的，就这两个好了，但是3`端有引物二聚体，可以用吗？第一个：
Current Oligo--------------------------------------------------------------------------------Sequen 关键词:[引物 引物二聚体]……
请高手帮我看看这两个引物，用PRIMER PREMIER搜索的，OLIGO验证的，就这两个好了，但是3`端有引物二聚体，可以用吗？第一个：
Current Oligo--------------------------------------------------------------------------------Sequence Length:
19Current Oligo (+ strand)5" AGA GCG AAA CAA GCA GAA C 3"Length:
59.8 °C (salt 1000.0 mM; oligo 100.0 pM)第二个：
Current Oligo--------------------------------------------------------------------------------Sequence Length:
19Current Oligo (+ strand)5" GGT CCT GAA GAA GAT TGC G 3"Length:
62.4 °C (salt 1000.0 mM; oligo 100.0 pM)deltaG (25 °C):
-36.4 kcal/mol3"deltaG
-10.5 kcal/molDegeneracy:
5.27 nmol/A260
31.5 μg/A260Current Oligo (- strand)5" CGC AAT CTT CTT CAG GAC C 3"P.E.#:
5.82 nmol/A260
33.8 μg/A260--------------------------------------------------------------------------------
Selected Primers--------------------------------------------------------------------------------New:435U19 Upper Primer5" GGT CCT GAA GAA GAT TGC G 3"Length:
62.4 °C (salt 1000.0 mM; oligo 100.0 pM)deltaG (25 °C):
-36.4 kcal/mol3"deltaG
-10.5 kcal/molDegeneracy:
427/4271/E:
5.27 nmol/A260
31.5 μg/A260New:499L18 Lower Primer5" GAT GCT CGG GAA GGT GAA 3"Length:
63.0 °C (salt 1000.0 mM; oligo 100.0 pM)deltaG (25 °C):
-36.0 kcal/mol3"deltaG
-6.7 kcal/molDegeneracy:
5.41 nmol/A260
30.9 μg/A260--------------------------------------------------------------------------------
Current Oligo Duplexes--------------------------------------------------------------------------------Current Oligo 19-mer [435]Current- Oligo: the most stable 3"-dimer: 2 bp, -3.6 kcal/mol5" GGTCCTGAAGAAGATTGCG 3"
3" GCGTTAGAAGAAGTCCTGG 5"Current- Oligo: the most stable 3"-dimer: 2 bp, -3.1 kcal/mol5" CGCAATCTTCTTCAGGACC 3"
3" CCAGGACTTCTTCTAACGC 5"Current- Oligo: the most stable dimer overall: 2 bp, -3.6 kcal/mol5" GGTCCTGAAGAAGATTGCG 3"
3" GCGTTAGAAGAAGTCCTGG 5"No stems longer than 2 bp.--------------------------------------------------------------------------------
Upper Primer Duplexes--------------------------------------------------------------------------------Upper Primer New:435U19The most stable 3"-dimer: 2 bp, -3.6 kcal/mol5" GGTCCTGAAGAAGATTGCG 3"
3" GCGTTAGAAGAAGTCCTGG 5"The most stable dimer overall: 2 bp, -3.6 kcal/mol5" GGTCCTGAAGAAGATTGCG 3"
3" GCGTTAGAAGAAGTCCTGG 5"No stems longer than 2 bp.--------------------------------------------------------------------------------
Lower Primer Duplexes--------------------------------------------------------------------------------Lower Primer New:499L18No 3"-terminal dimer formationThe most stable dimer overall: 2 bp, -3.6 kcal/mol
5" GATGCTCGGGAAGGTGAA 3"
: : || : :
3" AAGTGGAAGGGCTCGTAG 5"No stems longer than 2 bp.--------------------------------------------------------------------------------
Upper-Lower Duplexes--------------------------------------------------------------------------------Primers:
New:435U19
New:499L18The most stable 3"-dimer: 2 bp, -3.6 kcal/mol5" GGTCCTGAAGAAGATTGCG 3"
3" AAGTGGAAGGGCTCGTAG 5"The most stable 3"-dimer: 2 bp, -1.9 kcal/mol5" GGTCCTGAAGAAGATTGCG 3"
3" AAGTGGAAGGGCTCGTAG 5"The most stable dimer overall: 3 bp, -4.7 kcal/mol
5" GGTCCTGAAGAAGATTGCG 3"
3" AAGTGGAAGGGCTCGTAG 5"--------------------------------------------------------------------------------
Current Oligo Hairpin Stems--------------------------------------------------------------------------------No stems longer than 2 bp.--------------------------------------------------------------------------------
Upper Primer Hairpin Stems--------------------------------------------------------------------------------No stems longer than 2 bp.--------------------------------------------------------------------------------
Lower Primer Hairpin Stems--------------------------------------------------------------------------------No stems longer than 2 bp.--------------------------------------------------------------------------------
Current Oligo Composition--------------------------------------------------------------------------------Current Oligo 19-mer [435]Td = 62.4° [nearest neighbor method]Tm = 72.9° [%GC method]Tm = 58°
[2(A+T)° + 4(G+C)° method]Tm(RNA)[1M Na]
= 84.7° [%GC method]Tm(:RNA)[1M Na] = 79.0° [%GC method]A260/A280 = 1.92
[single strand]Mr
[one strand]Mr
= 11.7K [two strands]μg/OD
[ds]------------------------ Base
Number & %------------------------
4 [21.1%] A + T
9 [47.4%] G + C
10 [52.6%]--------------------------------------------------------------DNA Melting Temperature in VariousSalt and Formamide Concentrations [°C]--------------------------------------
50%--------------------------------------
40.4----------------------------------------------------------------------------Approximate Tm of the mismatched oligoMismatch Tm = Td - 1.2(% mismatch)°-------------------------------------- mism. #
Tm--------------------------------------
30.8----------------------------------------------------------------------------------------------------------------------
Upper Primer Composition--------------------------------------------------------------------------------Upper Primer New:435U19Td = 62.4° [nearest neighbor method]Tm = 72.9° [%GC method]Tm = 58°
[2(A+T)° + 4(G+C)° method]Tm(RNA)[1M Na]
= 84.7° [%GC method]Tm(DNA:RNA)[1M Na] = 79.0° [%GC method]A260/A280 = 1.92
[single strand]Mr
[one strand]Mr
= 11.7K [two strands]μg/OD
[dsDNA]------------------------ Base
Number & %------------------------
4 [21.1%] A + T
9 [47.4%] G + C
10 [52.6%]--------------------------------------------------------------DNA Melting Temperature in VariousSalt and Formamide Concentrations [°C]--------------------------------------
50%--------------------------------------
40.4----------------------------------------------------------------------------Approximate Tm of the mismatched oligoMismatch Tm = Td - 1.2(% mismatch)°-------------------------------------- mism. #
Tm--------------------------------------
30.8----------------------------------------------------------------------------------------------------------------------
Lower Primer Composition--------------------------------------------------------------------------------Lower Primer New:499L18Td = 63.0° [nearest neighbor method]Tm = 72.7° [%GC method]Tm = 56°
[2(A+T)° + 4(G+C)° method]Tm(RNA)[1M Na]
= 85.3° [%GC method]Tm(DNA:RNA)[1M Na] = 79.8° [%GC method]A260/A280 = 1.97
[single strand]Mr
[one strand]Mr
= 11.1K [two strands]μg/OD
[dsDNA]------------------------ Base
Number & %------------------------
3 [16.7%] A + T
8 [44.4%] G + C
10 [55.6%]--------------------------------------------------------------DNA Melting Temperature in VariousSalt and Formamide Concentrations [°C]--------------------------------------
50%--------------------------------------
40.2----------------------------------------------------------------------------Approximate Tm of the mismatched oligoMismatch Tm = Td - 1.2(% mismatch)°-------------------------------------- mism. #
Tm--------------------------------------
29.7----------------------------------------------------------------------------------------------------------------------
Upper Primer False Priming Sites--------------------------------------------------------------------------------Upper Primer - New:435U19
(positive strand)Priming efficiency of the perfect match is 427 (above the threshold)Not foundUpper Primer - New:435U19
(negative strand)Priming efficiency of the perfect match is 427 (above the threshold)Priming efficiency: 427 (above the threshold)5"(435) GGTCCTGAAGAAGATTGCG (453)3"
|||||||||||||||||||3"(435) ccaggacttcttctaacgc (453)5"--------------------------------------------------------------------------------
Lower Primer False Priming Sites--------------------------------------------------------------------------------Lower Primer - New:499L18
(positive strand)Priming efficiency of the perfect match is 429 (above the threshold)Not foundLower Primer - New:499L18
(negative strand)Priming efficiency of the perfect match is 429 (above the threshold)Not found--------------------------------------------------------------------------------
PCR--------------------------------------------------------------------------------Optimal Annealing Temperature: 53.0° (Max: 69.9°)--------------------------------------------------------------------
P.E.#--------------------------------------------------------------------Product
52.4Upper Primer
427/427Lower Primer
0/429--------------------------------------------------------------------Product Tm - Upper Primer Tm: 15.9Primers Tm difference: 1.4----------------------------------
Concentration----------------------------------Upper Primer
200.0 nMLower Primer
200.0 nMMonovalent Cation
50.0 mMFree Mg[2+]
0.7 mM----------------------------------Total Na[+] Equivalent: 155.8Comments:Terminal stability of the Upper Primer is too high.--------------------------------------------------------------------------------
Upper Primer Internal Stability--------------------------------------------------------------------------------Position: 435Length: 195" GGTCCTGAAGAAGATTGCG 3"--------------------------------------------------------------------------------
deltaG--------------------------------------------------------------------------------
10.5----------------------------------------------------------------------------------------------------------------------------------------------------------------
Lower Primer Internal Stability--------------------------------------------------------------------------------Position: 499Length: 185" GATGCTCGGGAAGGTGAA 3"--------------------------------------------------------------------------------
deltaG--------------------------------------------------------------------------------
6.7----------------------------------------------------------------------------------------------------------------------------------------------------------------
Internal Stability----------------------------------------------------------------------------------------------------------------------------------------------------------------
deltaG--------------------------------------------------------------------------------
10.5----------------------------------------------------------------------deltaG (25 °C):
-35.0 kcal/mol3"deltaG
-6.4 kcal/molDegeneracy:
4.88 nmol/A260
29.1 μg/A260Current Oligo (- strand)5" GTT CTG CTT GTT TCG CTC T 3"P.E.#:
6.33 nmol/A260
36.9 μg/A260--------------------------------------------------------------------------------
Selected Primers--------------------------------------------------------------------------------New:195U19 Upper Primer5" AGA GCG AAA CAA GCA GAA C 3"Length:
59.8 °C (salt 1000.0 mM; oligo 100.0 pM)deltaG (25 °C):
-35.0 kcal/mol3"deltaG
-6.4 kcal/molDegeneracy:
385/3851/E:
4.88 nmol/A260
29.1 μg/A260New:700L18 Lower Primer5" CCT CTG GCG AAG AAA CTC 3"Length:
59.5 °C (salt 1000.0 mM; oligo 100.0 pM)deltaG (25 °C):
-34.6 kcal/mol3"deltaG
-6.4 kcal/molDegeneracy:
5.92 nmol/A260
32.9 μg/A260--------------------------------------------------------------------------------
Current Oligo Duplexes--------------------------------------------------------------------------------Current Oligo 19-mer [195]Current- Oligo: no 3"-terminal dimer formationCurrent- Oligo: no 3"-terminal dimer formationCurrent- Oligo: the most stable dimer overall: 2 bp, -3.6 kcal/mol
5" AGAGCGAAACAAGCAGAAC 3"
3" CAAGACGAACAAAGCGAGA 5"No stems longer than 2 bp.--------------------------------------------------------------------------------
Upper Primer Duplexes--------------------------------------------------------------------------------Upper Primer New:195U19No 3"-terminal dimer formationThe most stable dimer overall: 2 bp, -3.6 kcal/mol
5" AGAGCGAAACAAGCAGAAC 3"
3" CAAGACGAACAAAGCGAGA 5"No stems longer than 2 bp.--------------------------------------------------------------------------------
Lower Primer Duplexes--------------------------------------------------------------------------------Lower Primer New:700L18The most stable 3"-dimer: 2 bp, -1.6 kcal/mol5" CCTCTGGCGAAGAAACTC 3"
3" CTCAAAGAAGCGGTCTCC 5"The most stable dimer overall: 2 bp, -3.6 kcal/mol
5" CCTCTGGCGAAGAAACTC 3"
3" CTCAAAGAAGCGGTCTCC 5"Hairpin: DG = 1.20 kcal/mol, Loop = 5 nt
5" CCTCTGG
C3" CTCAAAGAAG--------------------------------------------------------------------------------
Upper-Lower Duplexes--------------------------------------------------------------------------------Primers:
New:195U19
New:700L18No 3"-terminal dimer formationThe most stable 3"-dimer: 3 bp, -3.2 kcal/mol5" AGAGCGAAACAAGCAGAAC 3"
3" CTCAAAGAAGCGGTCTCC 5"The most stable dimer overall: 4 bp, -5.1 kcal/mol5" AGAGCGAAACAAGCAGAAC 3"
3" CTCAAAGAAGCGGTCTCC 5"--------------------------------------------------------------------------------
Current Oligo Hairpin Stems--------------------------------------------------------------------------------No stems longer than 2 bp.--------------------------------------------------------------------------------
Upper Primer Hairpin Stems--------------------------------------------------------------------------------No stems longer than 2 bp.--------------------------------------------------------------------------------
Lower Primer Hairpin Stems--------------------------------------------------------------------------------1. Duplex length = 3 DG = 1.2 kcal/ loop 5 nucleotides 3 TCT 5
|||13 AGA 11
5" CCTCTGG
C3" CTCAAAGAAG--------------------------------------------------------------------------------
Current Oligo Composition--------------------------------------------------------------------------------Current Oligo 19-mer [195]Td = 59.8° [nearest neighbor method]Tm = 70.8° [%GC method]Tm = 56°
[2(A+T)° + 4(G+C)° method]Tm(RNA)[1M Na]
= 81.0° [%GC method]Tm(DNA:RNA)[1M Na] = 74.7° [%GC method]A260/A280 = 2.34
[single strand]Mr
[one strand]Mr
= 11.7K [two strands]μg/OD
[dsDNA]------------------------ Base
Number & %------------------------
10 [52.6%]
5 [26.3%] A + T
10 [52.6%] G + C
9 [47.4%]--------------------------------------------------------------DNA Melting Temperature in VariousSalt and Formamide Concentrations [°C]--------------------------------------
50%--------------------------------------
38.3----------------------------------------------------------------------------Approximate Tm of the mismatched oligoMismatch Tm = Td - 1.2(% mismatch)°-------------------------------------- mism. #
Tm--------------------------------------
28.2----------------------------------------------------------------------------------------------------------------------
Upper Primer Composition--------------------------------------------------------------------------------Upper Primer New:195U19Td = 59.8° [nearest neighbor method]Tm = 70.8° [%GC method]Tm = 56°
[2(A+T)° + 4(G+C)° method]Tm(RNA)[1M Na]
= 81.0° [%GC method]Tm(DNA:RNA)[1M Na] = 74.7° [%GC method]A260/A280 = 2.34
[single strand]Mr
[one strand]Mr
= 11.7K [two strands]μg/OD
[dsDNA]------------------------ Base
Number & %------------------------
10 [52.6%]
5 [26.3%] A + T
10 [52.6%] G + C
9 [47.4%]--------------------------------------------------------------DNA Melting Temperature in VariousSalt and Formamide Concentrations [°C]--------------------------------------
50%--------------------------------------
38.3----------------------------------------------------------------------------Approximate Tm of the mismatched oligoMismatch Tm = Td - 1.2(% mismatch)°-------------------------------------- mism. #
Tm--------------------------------------
28.2----------------------------------------------------------------------------------------------------------------------
Lower Primer Composition--------------------------------------------------------------------------------Lower Primer New:700L18Td = 59.5° [nearest neighbor method]Tm = 72.7° [%GC method]Tm = 56°
[2(A+T)° + 4(G+C)° method]Tm(RNA)[1M Na]
= 85.3° [%GC method]Tm(DNA:RNA)[1M Na] = 79.8° [%GC method]A260/A280 = 1.86
[single strand]Mr
[one strand]Mr
= 11.1K [two strands]μg/OD
[dsDNA]------------------------ Base
Number & %------------------------
3 [16.7%] A + T
8 [44.4%] G + C
10 [55.6%]--------------------------------------------------------------DNA Melting Temperature in VariousSalt and Formamide Concentrations [°C]--------------------------------------
50%--------------------------------------
40.2----------------------------------------------------------------------------Approximate Tm of the mismatched oligoMismatch Tm = Td - 1.2(% mismatch)°-------------------------------------- mism. #
Tm--------------------------------------
26.2----------------------------------------------------------------------------------------------------------------------
Upper Primer False Priming Sites--------------------------------------------------------------------------------Upper Primer - New:195U19
(positive strand)Priming efficiency of the perfect match is 385 (above the threshold)Not foundUpper Primer - New:195U19
(negative strand)Priming efficiency of the perfect match is 385 (above the threshold)Priming efficiency: 385 (above the threshold)5"(195) AGAGCGAAACAAGCAGAAC (213)3"
|||||||||||||||||||3"(195) tctcgctttgttcgtcttg (213)5"--------------------------------------------------------------------------------
Lower Primer False Priming Sites--------------------------------------------------------------------------------Lower Primer - New:700L18
(positive strand)Priming efficiency of the perfect match is 418 (above the threshold)Not foundLower Primer - New:700L18
(negative strand)Priming efficiency of the perfect match is 418 (above the threshold)Not found--------------------------------------------------------------------------------
PCR--------------------------------------------------------------------------------Optimal Annealing Temperature: 54.3° (Max: 67.3°)--------------------------------------------------------------------
P.E.#--------------------------------------------------------------------Product
47.0Upper Primer
385/385Lower Primer
0/418--------------------------------------------------------------------Product Tm - Upper Primer Tm: 21.4Primers Tm difference: 0.2----------------------------------
Concentration----------------------------------Upper Primer
200.0 nMLower Primer
200.0 nMMonovalent Cation
50.0 mMFree Mg[2+]
0.7 mM----------------------------------Total Na[+] Equivalent: 155.8--------------------------------------------------------------------------------
Upper Primer Internal Stability--------------------------------------------------------------------------------Position: 195Length: 195" AGAGCGAAACAAGCAGAAC 3"--------------------------------------------------------------------------------
deltaG--------------------------------------------------------------------------------
6.4----------------------------------------------------------------------------------------------------------------------------------------------------------------
Lower Primer Internal Stability--------------------------------------------------------------------------------Position: 700Length: 185" CCTCTGGCGAAGAAACTC 3"--------------------------------------------------------------------------------
deltaG--------------------------------------------------------------------------------
6.4----------------------------------------------------------------------------------------------------------------------------------------------------------------
Internal Stability----------------------------------------------------------------------------------------------------------------------------------------------------------------
deltaG--------------------------------------------------------------------------------
6.4--------------------------------------------------------------------------------
回复:我觉得可以用，没什么问题的，3‘端就一个碱基配对，无甚大碍。回复:这种引物应该可以用的，我现在都不用软件设计引物，自行设计就可以，一般不会出现问题的。
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