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How to clone a gene with lots of splicing intermediates?

Genetics as it applies to evolution, molecular biology, and medical aspects.

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How to clone a gene with lots of splicing intermediates?

Postby polkadotshorts » Sat Nov 24, 2012 6:49 pm

I have been trying very very hard to clone the ligase 6 gene in Arabidopsis overexpress it. However, it has been proving very difficult due to the high amount of splicing intermediates.

I have so far used varying primers on the cDNA of arabidopsis in order to get some fragments that I could then ligate together. I have used proofreading polymerase, Taq, phusion, abgene, and I have then tried (and failed) to isolate, purify and sequence the fragment. The sequence results I get are always very low quality!

Does anyone have any ideas of what to do in such situations?

(It is my first time in a real university lab, so I am very inexperienced - therefore, nothing you say will be 'too simple'! :) please state the obvious, if needs be!)
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Postby polkadotshorts » Sat Nov 24, 2012 7:18 pm

Anyone?
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Postby JackBean » Sat Nov 24, 2012 8:07 pm

Hardly will someone answer in half hour.

Where do you want to express it? In what system? If you wanted to use eukaryotic system (yeast or best plants), you could clone genomic version including the introns and let the cell to solve your problem.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby JackBean » Sat Nov 24, 2012 8:08 pm

However in such case, you could never predict the outcome. It would vary based on the organism used and maybe also batch-to-batch.
http://www.biolib.cz/en/main/

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Re:

Postby polkadotshorts » Sat Dec 08, 2012 3:52 pm

JackBean wrote:Hardly will someone answer in half hour.

Where do you want to express it? In what system? If you wanted to use eukaryotic system (yeast or best plants), you could clone genomic version including the introns and let the cell to solve your problem.


Hello, thank you for your response :D sorry, this is my first post on this website, so I am not quite proficient when using it (hence my very late reply!).

I would want to overexpress it in arabidopsis seeds to observe germination rates under high stress conditions etc. I have suggested cloning it (with introns in) to my supervisor but he said that getting the intron-free clone is the most important part.

Moving on from that, I am trying to write some content about the alternative splicing nature of the ligase 6 gene from a gel image that I have taken. If you have the time, I would really appreaciate it if you could help me out! I am finding it all very difficult;
Each lane in the gel has cDNA that has been amplified with a set of primers that cut out an exon from the ligase 6 gene - so each lane should have one exon in it - however some lanes have 2 bands when they should theoretically have 1. I don't suppose you know what this says about the alternative splicing nature of the gene?

(I am terrible at explaining things, so please let me know if you'd need more information!)
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Postby JackBean » Mon Dec 10, 2012 6:10 pm

that would suggest there is some small alternative-intron in your exon, which can be thus split

why are you using primers only for one exon at a time?

Is your aim to clone all variants or are you fine with only some?
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Re:

Postby polkadotshorts » Mon Dec 10, 2012 7:12 pm

JackBean wrote:that would suggest there is some small alternative-intron in your exon, which can be thus split

why are you using primers only for one exon at a time?

Is your aim to clone all variants or are you fine with only some?



Well, we initially had 4 sets of primers. I was going to amplify 4 overlapping regions of the gene, run them on a gel, cut out the bands of expected size, purify them, and eventually sequence and ligate them together to generate the full length gene which I would then clone and amplify.

However, there were troubles with the isolation of the bands. I ran the experiment a number of times and didn't always get the same result (despite the exact same procedure and minimal scope for human error).

My supervisor suggested that this could be something to do with the splicing nature of the gene.... he suggested that we stop trying to clone the whole gene and look at the splicing nature of ligase 6 before we re-attempt any cloning or over-expression. In order to investigate the splicing nature of the gene, I created primers for each exon and generated a gel image. I should be able to identify all of the splice variants of the gene by using the gel image and observing the band sizes produced - the part that I am currently having the most difficulty.
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