'harmless' PCR test

[theodorn at medlib.georgetown.edu]

In article <gilbride-0812982013070001 at ppp-76-4.bu.edu>,
  gilbride at bu.edu (kevin gilbride) wrote:
> In article <01BE1D39.BAE713C0.MackayJF at bmnz.co.nz>, "MackayJF at bmnz.co.nz"
> <MackayJF at bmnz.co.nz> wrote:
>
> >
> > John
>
> Beta-Globin, structural protein found in all cells
> Primer 1, BG1 5'-AgC-ggg-AAA-TCg-TCg-gTg-3'
> Primer 2, BG2 5'-CAg-ggT-ACA-Tgg-TgC-3'
> 95º, 30s'
> 55º, 50s'
> 72º, 30s'
> 25-30 cycles 280 bp product

Do you mean beta-actin? (in any case, to amplify a piece of genomic DNA, it
wouldn't matter what cells you used)

Nick Theodorakis

Also, did you double check your primer sequence?
(see below)

> Primer 1, BG1 5'-AgC-ggg-AAA-TCg-TCg-gTg-3'

gb|U00039|ECOUW76  E. coli chromosomal region from 76.0 to 81.5 ...  36 
0.012 gb|AE000427|AE000427  Escherichia coli K-12 MG1655 section 317 o...  36
 0.012 dbj|D15072|KPNMOAR  Klebsiella aerogenes moaR gene for positive ... 
32  0.18 emb|Z66496|CEE04D5  Caenorhabditis elegans cosmid E04D5, complet... 
30  0.71 emb|Z83217|CEC10C6  Caenorhabditis elegans cosmid C10C6, complet... 
30  0.71 gb|M88485|PSETABA  Pseudomonas syringae tabA and tabB genes, com... 
30  0.71 gb|AC005558|AC005558  Drosophila melanogaster DNA sequence (P1s ... 
30  0.71 gb|AC004422|AC004422  Drosophila melanogaster (P1 DS08493 (D141)... 
28  2.8 gb|J04036|MUSB3RP  Mus musculus (clone p70ZN8) band 3-related pr... 
28  2.8 gb|AF087641|AF087641  Sinorhizobium meliloti ISRm102F34-1 putati... 
28  2.8 gb|U45885|RNU45885  Rattus norvegicus AE2 Cl-/HCO3- exchanger, v... 
28  2.8 gb|U63095|MSU63095  Mycobacterium smegmatis acetamidase regulato... 
28  2.8 gb|AC004280|AC004280  Drosophila melanogaster DNA sequence (P1 D... 
28  2.8 emb|Z95585|MTCY22G8  Mycobacterium tuberculosis H37Rv complete g... 
28  2.8 gb|U39357|OAU39357  Ovis aries beta actin mRNA, complete cds  28  2.8
dbj|D90901|D90901  Synechocystis sp. PCC6803 complete genome, 3/...  28  2.8
gb|U23177|CELC56G2  Caenorhabditis elegans cosmid C56G2  28  2.8
gb|U02295|BTU02295  Bos taurus Holstein beta-actin pseudogene, 5...  28  2.8
emb|Z81066|CEF17B5  Caenorhabditis elegans cosmid F17B5, complet...  28  2.8
emb|Z74410|MTCY251  Mycobacterium tuberculosis H37Rv complete ge...  28  2.8
emb|X66124|RMPLNFE  R.meliloti plasmid DNA for nfe locus  28  2.8
gb|J05166|RATBAND33E  Rat band 3 Cl-/HCO3- exchanger (B3RP2) mRN...  28  2.8
gb|AF025469|CELW09B6  Caenorhabditis elegans cosmid W09B6  28  2.8
gb|AF035422|AF035422  Ovis aries beta-actin mRNA, partial cds  28  2.8



gb|U00039|ECOUW76 E. coli chromosomal region from 76.0 to 81.5 minutes
             Length = 225419

 Score = 36.2 bits (18), Expect = 0.012
 Identities = 18/18 (100%), Positives = 18/18 (100%)


Query: 1     agcgggaaatcgtcggtg 18
             ||||||||||||||||||
Sbjct: 74028 agcgggaaatcgtcggtg 74045


 gb|AE000427|AE000427 Escherichia coli K-12 MG1655 section 317 of 400 of the
complete
            genome
            Length = 12853

 Score = 36.2 bits (18), Expect = 0.012
 Identities = 18/18 (100%), Positives = 18/18 (100%)


Query: 1    agcgggaaatcgtcggtg 18
            ||||||||||||||||||
Sbjct: 8886 agcgggaaatcgtcggtg 8903

etc...

> Primer 2, BG2 5'-CAg-ggT-ACA-Tgg-TgC-3'

Sequences producing significant alignments:  (bits)  Value

dbj|AB010266|AB010266  Mus musculus Creb-rp, Tnx and Cyp21 genes...  30  0.71
gb|AF030001|MMMHC29N7  Mus musculus major histocompatibility loc...  30  0.71
emb|AL022719|HS231L4  Human DNA sequence *** SEQUENCING IN PROGR...  30  0.71
gb|AC005156|AC005156  Homo sapiens PAC clone DJ1099C19 from 7q21...  30  0.71
dbj|D50028|CYISAA1  Carp mRNA for skeletal alpha-actin, complete...  28  2.8
gb|U67202|CFU67202  Canis familiaris beta-actin mRNA, partial cds  28  2.8
gb|M24113|CYIACTBA  Common carp (C.carpio) beta-actin gene, comp...  28  2.8
gb|M28424|HUMACTA1  Human beta-actin mRNA, partial cds	28  2.8
gb|U38850|FRU38850  Fugu rubripes alpha-skeletal actin1 gene, co...  28  2.8
gb|M22869|EMEACTINGA  Emericella nidulans gamma actin gene exons...  28  2.8
emb|X12452|DMACT87E  Drosophila melanogaster Act87E gene for actin  28	2.8
gb|M10277|HUMACCYBB  Human cytoplasmic beta-actin gene, complete...  28  2.8
gb|K02065|DROACT88G  D. melanogaster (mutant Ifm(3)7) act88F act...  28  2.8
emb|X59936|ABACT  A.bisexualis gene for actin  28  2.8 gb|U78026|NCU78026 
Neurospora crassa actin gene, complete cds  28	2.8 emb|X00306|RNAC03  Rat
mRNA fragment for cardiac actin  28  2.8 dbj|D85743|MLGMOCUMA2	Molgula
oculata DNA for actin, complete cds  28  2.8 dbj|D78191|MLGMOCCMAB  Ascidian
MoccMA1a pseudogene for actin, c...  28  2.8 gb|M18330|RATPKCDA  Rat protein
kinase C delta subspecies. >gi|2...  28  2.8 gb|M12866|MUSACSM	Mouse
skeletal muscle actin mRNA, complete cds.  28  2.8 emb|X63432|HSACTB 
H.sapiens ACTB mRNA for mutant beta-actin (be...  28  2.8 emb|X00351|HSAC07 
Human mRNA for beta-actin  28  2.8 emb|X16940|HSSMGA  Human mRNA for enteric
smooth muscle gamma-actin  28  2.8 gb|L07773|ECCACTNI  Echinococcus
granulosus actin 1 (actI), comp...  28	2.8 dbj|D89627|D89627  Oryzias
latipes mRNA for cytoplasmic actin Ol...  28  2.8 emb|X03470|XLACASR  Xenopus
laevis mRNA for alpha skeletal actin...  28  2.8 emb|X80130|RRALPHAAC 
R.rattus mRNA for alpha-actin cardiac protein  28  2.8 gb|M15501|MUSACTCM 
Mouse alpha-cardiac actin mRNA, 3' end.  28  2.8 gb|M12233|MUSACASM  Mouse
skeletal muscle alpha-actin mRNA.  28  2.8 gb|M64729|ABGACT2  Absidia glauca
actin (ACT2) mRNA, complete cds.  28  2.8 gb|AF025305|AF025305	Danio rerio
beta actin mRNA, complete cds  28  2.8 gb|U39357|OAU39357  Ovis aries beta
actin mRNA, complete cds  28  2.8 emb|X03766|MMACTASR  Mouse mRNA for
sceletal muscle alpha-actin ...  28  2.8 emb|X97615|HAACTA3B  H.armigera
actA3b gene  28  2.8 emb|X52605|ASACT403  Artemia mRNA for actin (clone
pArAct403)  28	2.8 gb|M18826|DROACT8F	D.melanogaster actin gene, complete
cds, loc...  28  2.8 gb|K00674|DROACT87E  D.melanogaster actin gene, complete
cds, lo...  28	2.8 gb|AF057040|AF057040  Danio rerio beta-actin mRNA,
complete cds  28  2.8 gb|U38848|FRU38848  Fugu rubripes beta-cytoplasmic
actin2 gene, ...  28  2.8 gb|K00667|DROACT5CX  D.melanogaster actin gene,
complete cds, lo...  28  2.8 gb|AF100986|AF100986  Penaeus monodon actin 1
(act1) mRNA, compl...  28  2.8 gb|AC004032|AC004032  Homo sapiens Chromosome
22q11.2 BAC Clone ...  28  2.8 gb|AF071908|AF071908  Bemisia argentifolii
actin gene, partial cds  28  2.8 dbj|D50659|D50659  Human gamma-cytoplasmic
actin (ACTGP9) pseudo...  28  2.8 emb|X97614|HAACTA3A  H.armigera actA3a gene
 28  2.8 dbj|D00653|HUMACTSG6  Homo sapiens gene for enteric smooth muscl... 
28  2.8 gb|AF079831|AF079831  Carassius auratus actin mRNA, partial cds  28 
2.8 emb|V00478|HSAC01  Human mRNA fragment encoding cytoplasmic acti...  28 
2.8 gb|U08283|OAU08283	Ovis aries actin mRNA, partial cds.  28  2.8
dbj|D14612|PRAACTIN  Crayfish mRNA for actin, partial sequence	28  2.8
gb|AC005781|AC005781  Homo sapiens chromosome 19, cosmid R33632,...  28  2.8
gb|U49644|BMU49644  Bombyx mori cytoplasmic actin (A4) gene, com...  28  2.8
dbj|D87740|D87740  Oryzias latipes mRNA for muscle actin OlMA1, ...  28  2.8
gb|U38961|FRU38961  Fugu rubripes alpha actin (alpha-cardiac act...  28  2.8
gb|U38960|FRU38960  Fugu rubripes alpha actin (alpha-cardiac act...  28  2.8
emb|X04669|XLACTCAG  Xenopus laevis cardiac actin gene	28  2.8
emb|X12525|XLACTA  Xenopus laevis sarcomeric alpha 3-actin gene  28  2.8
emb|X03469|XLACACR  Xenopus laevis mRNA for alpha cardiac actin ...  28  2.8
emb|X63502|XBACT3A  X.borealis gene for embryonic/larval alpha 3...  28  2.8
gb|M18830|DROACT88F  D.melanogaster actin gene, complete cds, lo...  28  2.8
gb|AF035422|AF035422  Ovis aries beta-actin mRNA, partial cds  28  2.8
gb|AF100987|AF100987  Penaeus monodon actin 2 (act2) mRNA, compl...  28  2.8
dbj|AB008817|AB008817  Ciona savignyi mRNA for muscle actin, com...  28  2.8
gb|L21915|MLGMUSCACT  Molgula citrina muscle actin mRNA, 3' end ...  28  2.8
gb|M12347|MUSACASA  Mouse skeletal alpha-actin gene, complete cds.  28	2.8
emb|X03767|MMACTCR  Mouse mRNA for adult cardiac muscle alpha-ac...  28  2.8
emb|X69078|HSSAMAA  H.sapiens mRNA for S-adenosylmethionine synt...  28  2.8
emb|X04507|BMACTA3  Bombyx mori cytoplasmic actin gene A3  28  2.8
emb|X02648|GG5ACT1  Chicken gene for type 5 nonmuscle actin >gi|...  28  2.8
emb|X60732|OCRNAASMA  O.cuniculus mRNA for alpha smooth muscle a...  28  2.8
gb|M87274|DROFLIACT  Drosophila simulans indirect flight muscle ...  28  2.8
gb|AC005146|AC005146  Homo sapiens 12q24.2 PAC RPCI1-157K6 (Rosw...  28  2.8
gb|AC004650|AC004650  Homo sapiens chromosome 5, P1 clone 207c2 ...  28  2.8
gb|U78027|HSU78027  Homo sapiens Bruton's tyrosine kinase (BTK),...  28  2.8
emb|X67536|HSP53RD  H. sapiens p53 responsive domain  28  2.8
gb|AC003923|AC003923  Drosophila melanogaster (P1 DS04219 (D36))...  28  2.8
gb|AF000240|AF000240  Gallus gallus monocarboxylate transporter ...  28  2.8
dbj|AB003910|AB003910  Fruitfly DNA for 88F actin, complete cds  28  2.8
gb|U49854|BMU49854  Bombyx mori cytoplasmic actin (A3) gene, com...  28  2.8
dbj|AB004047|AB004047  Human mRNA for beta-actin, complete cds	28  2.8




 dbj|AB010266|AB010266 Mus musculus Creb-rp, Tnx and Cyp21 genes for cAMP
response element
             binding protein-related protein, tenascin-X and steroid
             21-hydroxylase, partial cds and complete sequences
             Length = 67977

 Score = 30.2 bits (15), Expect = 0.71
 Identities = 15/15 (100%), Positives = 15/15 (100%)


Query: 1     cagggtacatggtgc 15
             |||||||||||||||
Sbjct: 19365 cagggtacatggtgc 19379


 gb|AF030001|MMMHC29N7 Mus musculus major histocompatibility locus class III
              region:butyrophilin-like protein gene, partial cds; Notch4,
              PBX2, RAGE, lysophatidic acid acyl transferase-alpha,
              palmitoyl-protein thioesterase 2 (PPT2), CREB-RP, a...
              Length = 201964

 Score = 30.2 bits (15), Expect = 0.71
 Identities = 15/15 (100%), Positives = 15/15 (100%)


Query: 1      cagggtacatggtgc 15
              |||||||||||||||
Sbjct: 150270 cagggtacatggtgc 150284

...


 gb|M28424|HUMACTA1 Human beta-actin mRNA, partial cds
           Length = 213

 Score = 28.2 bits (14), Expect = 2.8
 Identities = 14/14 (100%), Positives = 14/14 (100%)


Query: 1   cagggtacatggtg 14
           ||||||||||||||
Sbjct: 139 cagggtacatggtg 126

etc..

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