请教：100分的引物，到底如何

我初次接触PCR，准备做转基因后体内表到的半定量RT-PCR。用primer和oligo设计了一对引物，在primer上二个引物都打了100分，产物98分。但是有人说软件设计的引物有时候得分很高也会做不出来，所以想请各位高手帮忙看一下。:)

我对这对引物有两个疑问：
1 下游引物以AAT结尾是否不太好？
2 产物长度是213，是否太短了，跑胶不好看？

Selected Primers
------------------------------------------------------------------------------

748U19 Upper Primer

5' AAT GGT GGA CCG CAA CAA C 3'
Length: 19-mer
5' 748
Tm: 65.7 °C (salt 1000.0 mM; oligo 100.0 pM)
deltaG (25 °C): -37.6 kcal/mol
Degeneracy: 1
P.E.#: 442/442
1/E: 5.24 nmol/A260
31.0 μg/A260

942L19 Lower Primer

5' CCA AGG TAA CGC CAG GAA T 3'
Length: 19-mer
3' 942
Tm: 64.7 °C (salt 1000.0 mM; oligo 100.0 pM)
deltaG (25 °C): -38.5 kcal/mol
Degeneracy: 1
P.E.#: 453/453
1/E: 5.22 nmol/A260
30.9 μg/A260

PCR
------------------------------------------------------------------------------
Optimal Annealing Temperature: 54.5° (Max: 71.4°)

--------------------------------------------------------------------
Position Length Tm [°C] GC [%] P.E.#
--------------------------------------------------------------------
Product ----- 213 84.3 46.9 46.9
Upper Primer 748 19 70.3 52.6 442/442
Lower Primer 942 19 68.4 52.6 453/453
--------------------------------------------------------------------

Product Tm - Lower Primer Tm: 15.9
Primers Tm difference: 1.9

----------------------------------
Concentration
----------------------------------
Upper Primer 200.0 nM
Lower Primer 200.0 nM
Monovalent Cation 50.0 mM
Free Mg[2+] 0.7 mM
----------------------------------

Total Na[+] Equivalent: 155.8

Upper Primer Duplexes
------------------------------------------------------------------------------
The most stable 3'-dimer: 2 bp, -1.3 kcal/mol
5' AATGGTGGACCGCAACAAC 3'
|| : : ::
3' CAACAACGCCAGGTGGTAA 5'

The most stable dimer overall: 3 bp, -4.4 kcal/mol
5' AATGGTGGACCGCAACAAC 3'
||| :::
3' CAACAACGCCAGGTGGTAA 5'

No stems longer than 2 bp.

Lower Primer Duplexes
------------------------------------------------------------------------------
The most stable 3'-dimer: 2 bp, -1.5 kcal/mol
5' CCAAGGTAACGCCAGGAAT 3'
||
3' TAAGGACCGCAATGGAACC 5'

The most stable dimer overall: 2 bp, -3.6 kcal/mol
5' CCAAGGTAACGCCAGGAAT 3'
: || :
3' TAAGGACCGCAATGGAACC 5'

No stems longer than 2 bp.

Upper-Lower Duplexes
------------------------------------------------------------------------------
The most stable 3'-dimer: 2 bp, -1.3 kcal/mol
5' AATGGTGGACCGCAACAAC 3'
: : ||
3' TAAGGACCGCAATGGAACC 5'

The most stable 3'-dimer: 2 bp, -1.5 kcal/mol
5' AATGGTGGACCGCAACAAC 3'
|| : : : :
3' TAAGGACCGCAATGGAACC 5'

The most stable dimer overall: 3 bp, -5.0 kcal/mol
5' AATGGTGGACCGCAACAAC 3'
|||
3' TAAGGACCGCAATGGAACC 5'

Upper Primer False Priming Sites
------------------------------------------------------------------------------(positive strand)
Priming efficiency of the perfect match is 442 (above the threshold)

Priming efficiency: 41
5'(748) AATGGTGGACCGCAACAAC (766)3'
| | |||| |
3'(506) acctctcagggcgccgacc (488)5'

Priming efficiency: 36
5'(748) AATGGTGGACCGCAACAAC (766)3'
|||||| || || |
3'(1162) ctaccac-gggtccagtgg (1145)5'

(negative strand)
Priming efficiency of the perfect match is 442 (above the threshold)

Priming efficiency: 442 (above the threshold)
5'(748) AATGGTGGACCGCAACAAC (766)3'
|||||||||||||||||||
3'(748) ttaccacctggcgttgttg (766)5'

Priming efficiency: 118
5'(748) AATGGTGGACCGCAACAAC (766)3'
| | |||||| ||
3'(1) *tggcggagggcgtttctg (18)5'

Priming efficiency: 77
5'(748) AATGGTGGACCGCAACAAC (766)3'
|| ||||| ||||||
3'(898) ttgacacct--cgttgtgc (914)5'

Priming efficiency: 25
5'(748) AATGGTGGACCGCAACAAC (766)3'
| || || | | || |
3'(910) tgtgcatcttgagatggtc (928)5'

Priming efficiency: 12
5'(748) AATGGTGGACCGCAACAAC (766)3'
| |||||| | |
3'(1534) gttccacctcgtcaacagg (1552)5'

Lower Primer False Priming Sites
------------------------------------------------------------------------------ (positive strand)
Priming efficiency of the perfect match is 453 (above the threshold)

Priming efficiency: 453 (above the threshold)
5'(960) CCAAGGTAACGCCAGGAAT (942)3'
|||||||||||||||||||
3'(960) ggttccattgcggtcctta (942)5'

Priming efficiency: 63
5'(960) CCAAGGTAACGCCAGGAAT (942)3'
|||||| || | | ||
3'(308) ggttcctttccatccacta (290)5'

Priming efficiency: 34
5'(960) CCAAGGTAACG-CCAGGAAT (942)3'
||| || | ||| ||
3'(1208) ggtccccccaccggtactcc (1189)5'

(negative strand)
Priming efficiency of the perfect match is 453 (above the threshold)

Priming efficiency: 81
5'(960) CCAAGGTAACGCCAGGAAT (942)3'
|| | | | ||||||
3'(81) gggggcgtag-ggtcctgg (98)5'

Priming efficiency: 72
5'(960) CCAAGGTAACGCCAGGAAT (942)3'
| |||| ||||||
3'(581) gattccg-agcggtcaggg (598)5'

Priming efficiency: 45
5'(960) CCAAGGTAACGCCAGGAAT (942)3'
|| ||||||
3'(41) ggggaacgtgcggtgggag (59)5'

Priming efficiency: 44
5'(960) CCAAGGTAACGCCAGGAAT (942)3'
|| ||| || |||
3'(1412) tgtgtcatgtcgttccagg (1430)5'

Priming efficiency: 33
5'(960) CCAAGGTAA-CGCCAGGAAT (942)3'
|| ||||| || | |
3'(955) ggaaccattggccgacgact (974)5'

附件是mRNA序列