Primer sets for genetic libraries

[Wen-shue Wu]

Hi,everybody,

I wanna set up eukayotic rDNA genetic libraries of marine picophytoplankton. Eukaryotic 18S rRNA genes were amplified by PCR with eukaryotic-specific primers EUKA and EUKB, but failed. There is no products after PCR. I changed primer set that resulted in shorter fragments.
Should I do more try untill successful amplification with EUKA and EUKB, or accept other changed primer sets ? How can I get suitable primer sets for my samples? Use different sets and gather them?
Thanks in advance.

Wenshue

[Daniel Vaulot]

Several questions and suggestions :

- What kind of samples are your running, any chance of compound in sample interfering of PCR ?

- If you get success with shorter fragments, coul dbe that your DNA is sheared to small pieces during DNA extraction ?

- Also if you want really to look at picophytoplankton from filtered samples, might be best to use specific primers that are targetting photosynthetic groups (Chlrophyta, Haptophyta).

[PaulineBazin]

Hello everybody,
Iam a new user of molecular techniques and I am just starting my work, but my first goal is about yours : In summary I have to amplify the 18S rDNA genes from phytoplankton samples (GF/F filters, estuarine water) , generate clone banks and sequence them in order to evaluate the genetic diversity in the samples. For the beginning of my work (a test), I decided to use2 general eukaryotic primer sets found in literature: eukA-eukB (ex: Massana et al 2004) and euk328f-329r (ex: Moon-van der Stay et al 2000, Romari&Vaulot, 2004…). I managed to obtain bands at ≈ 1800pb, even if I these primers are not really specific. Indeed, other bands often appeared in the agarose gel, especially a recurrent upper band between 2000 and 2500pb, I don’t know what it means?!
 I think that if your samples are of the same kind as mine, it should work..  So, the problem can be due to the DNA extraction (integrity and purity of your extracted DNA), or maybe the PCR conditions. Finally maybe you can try euk328f-329r, it worked really well for me!


Pauline

[Daniel Vaulot]

Hi Pauline

Concerning the higher bands, this could be due to the fact that some eukaryotes have introns in the 18S rRNA gene which make therefore the amplified product larger thant the typical 1800 kb.  This is the case of forams I think but also some strains of groups such as Prasinophyceae may have introns.  This is the case of Bathycoccus which may or may not have an intron.  So better to clone everything and then analyze the sequences.

[Wen-shue Wu]

To Daniel,
-I am running nature samples (from marine stations),very normal.
-The community I really want to look at is Protist,exactlly.After this work,I may focus on specific groups using specific primers.

To Pauline,
-I just read papers you mentioned above.So I will have a try also using 328f-329r which worked well for you.Some other primer sets are on going.

Appriciate you advices very much!

Wenshue

[PaulineBazin]

Hi!
Thank you for informations, too. I am just sending my first samples (including the longer sequences (≈2500pb)) to the sequencing....

[Wen-shue Wu]

Hi,I amplified the sequence I wanted successfully today using Euk328f and Euk329r with beautiful bands.
Daniel, Pauline,Thank you very much.

Wenshue

[Lestag]

Hi, I'm presenting a poster at the ISME 13 next week that solves many of the primer issues here. We're not priming for PCR using DNA, but instead making total cDNA (mostly ribosomal whatever the organism) and tagging with a transposon that seeks out double-stranded regions in cDNA. Ribosomal rRNA (cDNA) has lots of secondary structure, as you know. The tagged cDNA fragments are 'lightly' amplified and can be next-gen sequenced. The result with a small number of pond cDNAs is a staggering variety of organisms, eukaryote and bacterial. The sequence reads include the small AND large subunit rRNAs, with coverage across most of the transcripts. Come see the poster if in Seatlle- it'll be on Monday pm under Les Hoffman.