pcr doesnt show any band for DNa walking - (Aug/11/2009 )

i have some questions...im doing genomic pcr to design 3 sets of primer to be used for DNA walking Kit Seegene..

1. im using gradient pcr for every primer ive tried but the result was so weird...when i view my gel no band can be seen, also no smearing at all..why?

2. sometimes i got only pcr product with size 150bp only...how to optimize the mixture and parameter of the pcr to get good band?

3. any suggestion on how to do dna walking?

please help me...

-hazrina-

Dear Hazrina

First of all, how long is your gene and what is your expected size of your PCR product?

I am a bit confuse that you have said your gradient PCR was no band, but then you said you got 150bp....

Adrian
p/s: I got a friend in Malaysia, Sarawak and her name ia also Hazrina....

-adrian kohsf-

dear adrian

this is me..hehehe...im having trouble with my project..

im doing dna walking for sago gene...how to know our expected size?yalah....sometime i got band for my gradient...but hard to get...the most was only 150bp..but that is not enogh for dna walking...do u know anything about dna walking?please advise me...also on intron exon...really cant recall although i read info on that...huhuhuh...

-hazrina-

hazrina on Aug 16 2009, 12:03 AM said:

Hi Haz,
For your sago gene, first you have to determine which sago gene you are looking for.
From your post, it seems that you are trying to get some gene from RNA. I assume you had got your cDNA by now. No need to afraid for the introns as your mRNA i suppose is a "mature" RNA, which had done splicing.

I suppose your experiment design was: 1)compare differentially expressed genes by RT-PCR for your all RNA using Random oligos. 2) GEL-excise the "different" bands and clone into vector and then sequencing. 3) find the full gene in native form (before splicing)

And if I were correct you are now stuck in the 3rd step.

I'm not very good in genome walking, but I would suggest you to use inverse PCR approach to get at least some portion of the genes , and try to work from here.

Also, if your size is 150 bps (make sure is not primer dimers)...I personally think you just need to sequence it rather than "walking" it.

I guess you still got my email. Just in case you dont have it, pm me.

p/s: how's everyone there? Say hello for me to Dr Hasnain and our mates there.

-adrian kohsf-

adrian,
i have looked at the link that u had given to me...dont u think that inverse PCR is similar to manual DNA walking?

now im not doin any cdna work..only the genomic dna...i need to do two works which is using manual n kit to get the promoter for adh gene in sago.

the problem now is that every time i do genomic pcr that is gonna be used to design three new primer...i got the useless sequence once i sent for sequencing...no adh gene is hit...so i cant create new primer for the kit to be used...

second...im doing also the manual way...which is re n ligate the dna...similar to the link you gave me..then..after ligation...i need to do two pcr..first is reverse primer with the adapter primer..once i get the band from the first cycle pcr..then i have to proceed to second cycle using reverse and nested primer...somehow...i got the band and had been sent for sequencng..the problem now is to analyze the result...how to know that wat i get the the right promoter for adh gene?is it the location of the intron exon, the tata boz, the gata, the cpg island or the open reading frame...how to know?

i dont have ur email..ive deleted my friendster...hehehe...

-hazrina-

Dear Haz,

I'm sorry I do not have wide experiences in plants...just try to share my thought.

First of all, i think is best to make sure you got your adh gene first before move into finding the promoter.

ChengCheng, (Dr Hairul MSc Std) did talk to me and we had discussed about finding promoter for sago genes as we meet in the recent 18 MSMBB conference.
Yes, we did talk about the inverse PCR and as well adapter primers. Since she point out the Sago palm is near to the Oil palm, why not you try to do sequence pairwise alignment with sago palm (adh??) promoter? You might able to guess the region. Also, we did agree that try to use oil palm forward primers with your sago reverse primer, you might be able to get the whole promoter out. since the promoter would not too far from the open reading frame, I guess if you can get roughly 50bases before your ORF is good enough. but however, according to Morton et al 1996 (PNAS article below), there is mention Potential TATA boxes for RNA polymerase binding are found 194 and 88 bases upstream of the start codon in the palm adhB sequence....

Here is an article at year 1984 on how people manage to get full length adh gene, quite detail.

http://www.pubmedcentral.nih.gov/articlere...ubmedid=6328449

In simple, they use RE to digest the whole genome of maize, run gel, use the partial adh gene which had cloned into the vector (radioactive), blotting it and compare, gel isolation of digested gene, clone into vector. sequence it.... imagine that ppl manage to do such in those days while we only learn all these lately...

Links below might be useful for you just in case you dont have it:

www.pnas.org/content/93/21/11735.full.pdf
http://www.pubmedcentral.nih.gov/articlere...;tool=pmcentrez
http://www.pubmedcentral.nih.gov/articlere...gi?artid=553349
http://palmoilis.mpob.gov.my/Netacgi/nph-b...&SECT5=PGEN

Tell me how you think.
^-^

-adrian kohsf-

Since she point out the Sago palm is near to the Oil palm, why not you try to do sequence pairwise alignment with sago palm (adh??) promoter? You might able to guess the region. Also, we did agree that try to use oil palm forward primers with your sago reverse primer, you might be able to get the whole promoter out. since the promoter would not too far from the open reading frame, I guess if you can get roughly 50bases before your ORF is good enough. but however, according to Morton et al 1996 (PNAS article below), there is mention Potential TATA boxes for RNA polymerase binding are found 194 and 88 bases upstream of the start codon in the palm adhB sequence....

what do u mean by oil palm?i need to find the adh sequence of oil plam and align it wit my sequence is it?what is oil palm forward primer?so blur....huhuhuuh...

-hazrina-

Hi Haz,

Oil palm in malay is called "Kelapa sawit". MPOB had done quite lots of work regarding this. Try to search around. Align the genes as well as with the promoter region and probably you "might guess out" the region you want.

Oil palm forward primer....I guess you had to ask around and find out yourself whether there is available. But I think is in one of the article I suggested you to go through.

Good luck.

-adrian kohsf-

Hello,

I have to do my primer walking on one gene (length of my cDNA 3000bp). Whole gene is covered with 11 amplicons approx. 300bp. For my primer walking I choose forward primer from amplicon 1. and reverse primer from amplicon 11.
I did my PCR with one of the lowes temperature (I made my choice by looking at the temperatures from other amplicons on that gene).
And when I put my sample on gel I got no band. I use o,8% agaraose gel in TBE buffer (everything fresh and new). I also made my PCR reaction in volume 50ul, and after PCR I add 10ul of loadding buffer.

Any ideas, why I got no bands?

Thanks a lot for your help.

-Hellena4-