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[Arabidopsis] Fwd: Re: chloroplast transformation vector

Patricia León via arab-gen%40net.bio.net (by patricia from ibt.unam.mx)
Wed Oct 12 16:16:26 EST 2016




-------- Mensaje reenviado --------
Asunto: 	Re: chloroplast transformation vector
Fecha: 	Wed, 12 Oct 2016 14:14:02 -0400
De: 	Myat Lin <mtl84 from cornell.edu>
Responder a: 	mtl84 from cornell.edu
Para: 	Patricia León <patricia from ibt.unam.mx>
CC: 	Maureen Hanson <mrh5 from cornell.edu>



Hi Patricia,

You are on the right track about getting DNA from a piece of leaf and 
putting the shoots to soil to get seeds.

I have attached a very nice protocol that I found online for DNA 
extraction. The main difference is that I don't add PVP to the CTAB buffer.

For restriction enzyme analysis, I have attached a PDF file to 
illustrate how it works. For your BJF locus, I use XhoI enzyme which 
gives a DNA fragment of about 3 kbp in the wild-type chloroplasts. My 
probe is downstream of the insertion site. Then, I will check if there 
is any XhoI site in the inserted DNA (transgenes) and calculate the size 
of the new DNA fragment digested by XhoI that can be detected by the 
probe. For southern blot protocol, I generate the DIG-labeled probe and 
follow the protocol by Roche Life Science. Let me know if you have any 
specific question.

Myat



On Tue, Oct 11, 2016 at 8:46 PM, Patricia León <patricia from ibt.unam.mx 
<mailto:patricia from ibt.unam.mx>> wrote:

    Hi Myat and Maureen,
    Just a brief report. We have already plants ith roots and our plan
    is to take a small leaf from them to make DNA and put them in soil
    to get seeds. Does this sounds correct. Also Myat I wonder if it
    would be possible to get your protocol for DNA preparation and a
    little bit more of the details about how do you decide the
    restiction and the probes.
    Best regards,
    Patricia

    El 9/19/16 a las 11:42 AM, Myat Lin escribió:
>     Hi Patricia,
>
>     I use MS agar medium (the same one used to grow seedlings for
>     bombardment) but with 500 mg/L spectinomycin for rooting the
>     transformants. I have attached a photo of a sample shoot and a
>     magenta box that we use to root the shoots. After 2-3 weeks when
>     the shoots have grown a bit more, you can extract DNA from a small
>     leaf tissue from each shoot to check whether they are homoplasmic.
>     The best way to check is to digest each DNA with a restriction
>     enzyme that will give different fragment size in the region of
>     interest and then (Southern) blot them with a DNA probe for that
>     region. I synthesize a DIG-labeled DNA probe for each region that
>     I want to check. Sometimes, it might be possible to check with
>     PCR. But I found that many of the chloroplast regions have at
>     least a copy in the nuclear genome and therefore PCR is not able
>     to distinguish the homoplasmic shoots from the heteroplasmic ones.
>
>     I will check my plamids and find a suitable one for you to
>     introduce two genes.
>
>     Myat
>
>
>
>     On Mon, Sep 19, 2016 at 11:40 AM, Patricia León
>     <patricia from ibt.unam.mx <mailto:patricia from ibt.unam.mx>> wrote:
>
>         Dear Myat,
>         I hope you are doing well. Here plants are continuing growing
>         in the 2 selection and I think we need to root them already.
>         We have several regenerants from the two independent events. I
>         would like to bother you if you could let me know how do you
>         proceed in this step? How big the plants you take and in which
>         media do you root tme. Also we will need to proceed to check
>         the transformants. I was thinking to do it through PCR. Is
>         this possible?. Finally I would like to ask you again if you
>         could send the plasmid to introduce the two genes.
>         Thank for the help and best wishes,
>         Patricia
>
>         El 8/11/16 a las 9:53 AM, Myat Lin escribió:
>>         Hi Patricia,
>>
>>         I have attached the sequence of a plasmid where I inserted
>>         TrbcLAt-IEE-SD-ccmO after the first gene ccmK2. I am out of
>>         town right now. I can send you the plasmid when I get back on
>>         Aug 22. In the meantime, if you have any question about how
>>         to introduce the second gene, please let me know.
>>
>>         Best wishes,
>>
>>         Myat
>>
>>
>>         On Thu, Aug 11, 2016 at 1:17 AM, Patricia León
>>         <patricia from ibt.unam.mx <mailto:patricia from ibt.unam.mx>> wrote:
>>
>>             Hi Myat,
>>             Regarding the constructs. That is exactly what I want to
>>             do. However I am not sure where to get the IEE-SD
>>             sequences. I have now the construct with the DXS that we
>>             cloned into the Nhe1 and Not sites in the pBNG1 vector.
>>             If I understand correct I would have to do primers for
>>             the IEE-SD sequence but first I would need if possible
>>             the sequence and if you have a vector with these
>>             sequences to amplify from there. I will appreciatte any
>>             imput and I am also checking the Occhialini paper.
>>             Regards,
>>             Patricia
>>             El 8/8/16 a las 8:16 PM, Myat Lin escribió:
>>>
>>>             Hi Patricia,
>>>
>>>             Two shoots is a pretty good result for the first
>>>             attempt. Yes, you need to cut these two shoots into ~
>>>             2-4 mm2 pieces and transfer them to the same selection
>>>             medium. Only put 3 pieces in each plate as they will
>>>             grow much bigger. You can have at least two plates for
>>>             each of the two shoots. You can use the remaining parts
>>>             of the shoots to check for the presence of your gene by
>>>             pcr. When the pieces from the second round grow into
>>>             shoots after 4-6 weeks , you need to extract DNA and run
>>>             southern blot to check whether the shoots are homoplasmic.
>>>
>>>             If you want to express two genes simultaneously from the
>>>             chloroplast genome, one way is to insert
>>>             terminator-IEE-SD between the two genes where IEE stands
>>>             for intercistronic expression element and SD stands for
>>>             shine dalgarno sequence. Please look at our 2016 plant
>>>             journal paper ( occhialini et al) for more detail on
>>>             these DNA sequences. In that paper, I used overlapping
>>>             pcr to join Terminator-IEE-SD with the second gene. Then
>>>             you can insert terminater-IEE-SD-gene2 by ligating it
>>>             into a restriction site right after your first gene.
>>>             Please note that the expression level of the second gene
>>>             is usually lower than the first gene in this
>>>             arrangement. If you need anything specific, please let
>>>             me know.
>>>
>>>             Best regards,
>>>
>
>




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