Cloning of ITS region - (Apr/10/2011 )

hi,

I am actually having a big problem in cloning the ITS region (800 bp) of a basidiomycete fungi into the pGEM-T Easy Vector (3015 bp). I have tried many times but yet I fail. I am using the protocol provided by the manufacturer (Promega). I have tried optimizing but still failed. After my PCR (ITS Region Amplification), i proceed to DNA Purification using MEGAquick-spin to get eluted DNA. Than, i proceed to ligation. For the standard reaction, i use 5uL of 10x Rapid Ligation buffer, 1uL of Vector (50ng), PCR product ( approximately 5uL depending on the intensity of the band obtained from purification of PCR products), 1 uL of T4 DNA Ligase (3 Weiss unit). I incubate overnight at 4C. The next day, i perform transformation. I thaw DH5a E.coli competent cells on ice. I spine briefly the ligation reaction before inserting into 100 uL DH5a into the tube. Incubate at 4C for 30min, heat shock at exactly 42C for 1 min and chill on ice for 2 min. Than i add 900 uL prewarmed LB Broth, incubate for 2 hours in thermoshaker (550 rpm at 37C). After that, i spin 13000 rpm for 1 min, discard about 800uL of supernatant to concentrate the cells. I re-suspend the cells and spread them on LB/Amp./IPTG/X-Gal plate and incubate at 37C for 16 hours. The very next day, when i observe, i only get to see white colonies but no blue colonies are seen. But when the white colonies are screened for inserts in PCR Colony, no bands are observed. I did plate those white colonies in LB/Amp plate to verify if they are false positive colonies, but they grew well in the LB/Amp. plate. I do not understand, when they can grow well in the replicate LB/Amp. plates, why than they do not have inserts. What can i do to get inserts in the colonies and successfully identify them in the PCR colony. PLZ HELP.

Advice and comments will be most appreciated. Thank you in advance.

Lizrene

If you just need to identify the fungus, just do direct sequencing will do. There is no need for you to clone it into pGEM-T.
Also, GEL-excise the band, do not just do column cleaning of PCR product.

Please check for contamination in your DH5a competent cells. Check your antibiotics plate, it might be too old...

Just to check if the obvious questions in your protocol are correct:
You have run the PCR product on a gel, and know that there is a band of the correct size.
The ligation is done in a 50 ul final volume (I assume, since you are using 5 ul of a 10x buffer).
The 4C incubation sounds a little low. I would incubate at 16C or room temperature for a while.
How are your preparing your vector?
How much of the ligation mixture are you adding to your cells? 2-5 ul should be used (more is inhibitory).
Two hour incubation may overgrow non-transformed cells. I would cut this back to 1 hour.
I would strongly recommend a control transformation of plasmid that was lacZ positive. It sounds as if your plates may not actually have X-Gal. I would also use S-Gal (Sigma) plates instead, since the phenotype is much more easily visualized. Blue colonies on X-gal plates are often not blue immediately. It helps to put them at 4C for a few hours, which tends to make the blue visible.

I agree with Adrian, you can alternatively sequence directly from the purified PCR product after gel purification, using either of the two primers used for the PCR.

hi there,

Thank you for ur advice n comments. I shall try the direct sequencing to identify the species. But will it give a good result just as pGEM-T cloning.

If you are using ITS1, ITS4, and every steps that was done correctly, from my experience it is fine.

Hello Lizrene,

My idea is the same with those of Adrian and Phage434.
Because ITS regions are located between rDNAs which are well conserved, you can design (a) good primer pair(s) to amplify and sequence ITSs directly without taking time and errors from cloning.
Your cloning protocol also seems not to be optimized as Phage434 pointed out.
Good luck

Hi,,

Thanks phucvn. Shall try on the direct sequencing of the ITS region.