headers
Cathal Garvey | 6 Jul 2011 14:26
Picon
Gravatar

Long-amplicons and Taq

(Crossposting from Google+)
I have a plasmid that's, say, 3.7kb long, and I need to amplify it with taq
polymerase (because that's what I have, and I'm too poor to afford better
right now). It's the length that's the main problem, although challenges
relating to the specifics of how to amplify it are also troubling me.

Specifically, I need to amplify a ~3.6kb plasmid *from another cloning
vector* as a linear molecule such that either amplicon end is complementary*
to the other, because this will enhance B.subtilis' ability to take up the
DNA and recombine it into a ring-shaped plasmid once more..or at least
enhance the plasmid's likelihood of copying itself successfully into a
ring-shaped plasmid by rolling-circle-replication before it is lost.

Taq seldom amplifies longer than 2kb, but there are voodoo ways to make it
do so. Among these voodoo methods are alternate buffers (apparently removing
potassium salt can improve matters?), different cycling conditions, and
different setup of the reaction.
I gather one of the big limiting factors is mismatches; taq extends
piecemeal, and if it detaches at a mismatch then there's no 3' end bound to
the template for the next enzyme to bind. So any method that enhances enzyme
fidelity should also enhance extension length.

Does anyone out there have tips on how to reliably achieve up to 4kb
amplification with bog-standard taq? I have a limited ability to make my own
buffers, and I can play with Magnesium and NTP concentrations easily enough.

*Anyone with information on how much complementarity is enough to induce
recombination in B.subtilis in this case? DNA is generally minced into
single-stranded DNA on uptake, and either end may suffer "nibbling" by
nucleases before circularising stably, so I'm imagining primers with a
(Continue reading)

Permalink | Reply | 0 comments |
headers
mnr mnr | 6 Jul 2011 19:23
Picon

Blunt end cloning

Hi all.

I have tried cloning a potential toxic gene about 1.2 kbp into E.coli with
T7 promoter. After two months, I am still not successful. I am now plannig
to do cloning into pUC18 vector before subcloning into few other vectors
with different promoters. I have limited available restriction sites so that
I don't have to redesign PCR primers. Is it feasible to do blunt-end cloning
with Phusion polymerase into non-dephosphorylated vectors? Will I only be
getting empty vectors after transformations?

Thank you.

Matt
Permalink | Reply | 3 comments |
headers
WS | 6 Jul 2011 23:49
Picon
Favicon

Re: Blunt end cloning

Hi Matt,

does that mean that you don't get any clone with an insert at all or
"just" no clone that has a correct insert (full length, no deletions,
no frameshifts etc.)?
pUC18 is promoterless, right?

As you suspect inherent toxicity, do you have any idea what kind of
toxicity it is? Will you need that toxic element or may you make it
"innocent" by some sort of AA substitution / motif deletion? Can you
possibly switch to another host like some sort of phage, YAC, (really
safe!!!) virus etc?

Wo

On Jul 6, 7:23 pm, mnr mnr <mnr...@...> wrote:
> Hi all.
>
> I have tried cloning a potential toxic gene about 1.2 kbp into E.coli with
> T7 promoter. After two months, I am still not successful. I am now plannig
> to do cloning into pUC18 vector before subcloning into few other vectors
> with different promoters. I have limited available restriction sites so that
> I don't have to redesign PCR primers. Is it feasible to do blunt-end cloning
> with Phusion polymerase into non-dephosphorylated vectors? Will I only be
> getting empty vectors after transformations?
>
> Thank you.
>
> Matt
(Continue reading)

Permalink | Reply | 3 comments |
headers
mnr mnr | 7 Jul 2011 01:57
Picon

Re: Blunt end cloning

Hi Wo.

It is a outer membrane protein, toxicity is probably at expression level. I
get inserts with random deletion that I can not use for functional assays.

Before I try other expression systems, I want to try other E. coli vectors
first. Am I going in the right direction?

On Wed, Jul 6, 2011 at 10:49 PM, WS <novalidaddress@...> wrote:

> Hi Matt,
>
> does that mean that you don't get any clone with an insert at all or
> "just" no clone that has a correct insert (full length, no deletions,
> no frameshifts etc.)?
> pUC18 is promoterless, right?
>
> As you suspect inherent toxicity, do you have any idea what kind of
> toxicity it is? Will you need that toxic element or may you make it
> "innocent" by some sort of AA substitution / motif deletion? Can you
> possibly switch to another host like some sort of phage, YAC, (really
> safe!!!) virus etc?
>
> Wo
>
>
>
>
> On Jul 6, 7:23 pm, mnr mnr <mnr...@...> wrote:
> > Hi all.
(Continue reading)

Permalink | Reply | 2 comments |
headers
Irit Rappley | 7 Jul 2011 03:36
Picon
Favicon

Re: Blunt end cloning

If your problem is inserts with random deletions, have you double-checked that your PCRs and ligations are
giving you correct sequences? You could try using a high-fidelity polymerase, if you don't already use one.

Do you think the deletions are happening upon expression and are due to the toxicity? If so, then you could
try growing your E.coli at 30C or even 20C during (longer than usual) induction because the slower protein
synthesis allows for better error correction. If not, then it seems to me that you have 2 separate issues
(cloning the correct sequence, and subsequent toxicity upon expression). 

Why do you think that the problem is with your vector?

Irit

On Jul 6, 2011, at 4:57 PM, mnr mnr wrote:

> Hi Wo.
> 
> It is a outer membrane protein, toxicity is probably at expression level. I
> get inserts with random deletion that I can not use for functional assays.
> 
> Before I try other expression systems, I want to try other E. coli vectors
> first. Am I going in the right direction?
> 
> 
> On Wed, Jul 6, 2011 at 10:49 PM, WS <novalidaddress@...> wrote:
> 
>> Hi Matt,
>> 
>> does that mean that you don't get any clone with an insert at all or
>> "just" no clone that has a correct insert (full length, no deletions,
>> no frameshifts etc.)?
(Continue reading)

Permalink | Reply | 0 comments |
headers
WS | 7 Jul 2011 07:59
Picon
Favicon

Re: Blunt end cloning

Hi Matt,

As Irit suggested, growing your bugs at different temperatures is an
option that is possibly easy to test and quite straightforward.

I don't think that the vector is the problem (as long it has a
promoter). Your protein simply might disturb membrane integrity or do
some bad signaling. Thus, you'll select for clones with non-functional
(at least: non-toxic) protein.

Do you have any inducible vectors, eg one of those that used for blue-
white selection? Maybe you can get hold of an E.coli strain that is
suitable for blue-white screening (eg XL1-blue, check the tech section
in the NEB catalogue or the internet). While growing the bacteria,
include glucose in the medium for efficient repression. Then you may
induce expression with galactose. In that scenario, you should at
least get some protein expression before your bacteria die.

Are there any 'inhibitors' that may interfere with your protein's
signaling pathways (if any) that you could add to the growth medium?

If your protein has one or more known partner(s), you also might try
co-expression. Cell-free expression systems might yield protein but
may lack any posttranslational processing (you might add something
like a His-tag for recovery). Before you switch to e.g. yeast, check
for homologous proteins first by BLASTing your gene of interest to
minimize the risk of running into the same problem again.

HTH
(Continue reading)

Permalink | Reply | 0 comments |
headers
Cathal Garvey | 7 Jul 2011 13:16
Picon
Gravatar

Re: Blunt end cloning

I'm confused; why not try to clone the sequence into a vector *without* a
promoter first? That way you can determine whether the gene's toxicity is
all that prevents you from getting a successful transformed culture.

Alternatively, clone it with an inducible promoter. That way you can try to
establish a stable clone, and then induce expression later to test for
toxicity. It is possible that by tuning the expression with different
amounts of induction, you can find a threshold that does not kill the cells
and allows you to produce some of the gene product.

If your gene *is* toxic and you persist in trying to clone it with T7,
you'll have a hard time. Any successful transformants will be selected
against immediately, leaving only those with partial deletions or mutations
that render the gene inoperable. Try it one piece at a time and it will be
easier to isolate the problem; cloning, transformation or expression.

On 7 July 2011 00:57, mnr mnr <mnr475@...> wrote:

> Hi Wo.
>
> It is a outer membrane protein, toxicity is probably at expression level. I
> get inserts with random deletion that I can not use for functional assays.
>
> Before I try other expression systems, I want to try other E. coli vectors
> first. Am I going in the right direction?
>
>
> On Wed, Jul 6, 2011 at 10:49 PM, WS <novalidaddress@...> wrote:
>
> > Hi Matt,
(Continue reading)

Permalink | Reply | 0 comments |
headers
chovek69 | 7 Jul 2011 13:47
Picon

Re: Long-amplicons and Taq

On Jul 6, 3:26 pm, Cathal Garvey <cathalgar...@...> wrote:
> (Crossposting from Google+)
> I have a plasmid that's, say, 3.7kb long, and I need to amplify it with taq
> polymerase (because that's what I have, and I'm too poor to afford better
> right now). It's the length that's the main problem, although challenges
> relating to the specifics of how to amplify it are also troubling me.
>
> Specifically, I need to amplify a ~3.6kb plasmid *from another cloning
> vector* as a linear molecule such that either amplicon end is complementary*
> to the other, because this will enhance B.subtilis' ability to take up the
> DNA and recombine it into a ring-shaped plasmid once more..or at least
> enhance the plasmid's likelihood of copying itself successfully into a
> ring-shaped plasmid by rolling-circle-replication before it is lost.
>
> Taq seldom amplifies longer than 2kb, but there are voodoo ways to make it
> do so. Among these voodoo methods are alternate buffers (apparently removing
> potassium salt can improve matters?), different cycling conditions, and
> different setup of the reaction.
> I gather one of the big limiting factors is mismatches; taq extends
> piecemeal, and if it detaches at a mismatch then there's no 3' end bound to
> the template for the next enzyme to bind. So any method that enhances enzyme
> fidelity should also enhance extension length.
>
> Does anyone out there have tips on how to reliably achieve up to 4kb
> amplification with bog-standard taq? I have a limited ability to make my own
> buffers, and I can play with Magnesium and NTP concentrations easily enough.
>
> *Anyone with information on how much complementarity is enough to induce
> recombination in B.subtilis in this case? DNA is generally minced into
> single-stranded DNA on uptake, and either end may suffer "nibbling" by
(Continue reading)

Permalink | Reply | 5 comments |
headers
Jonas Danielson | 8 Jul 2011 10:54
Picon
Picon

Re: Long-amplicons and Taq


Hi!

I don't think your main problem will be the length of the amplicon, but rather that you are extremely likely
to get errors, on average a 4kb product, when run for 20 cycles will have 2 errors, and finding a clone with no
errors will be almost impossible. If this is not a problem for you, fine go on with Taq, but otherwise,
change to something like Phusion because the extra money you will spend on the enzyme you will easily save
on not having to sequence that many clones (and it will save you some time too).

/jonas

On Jul 7, 2011, at 13:47 PM, chovek69 wrote:

> On Jul 6, 3:26 pm, Cathal Garvey <cathalgar...@...> wrote:
>> (Crossposting from Google+)
>> I have a plasmid that's, say, 3.7kb long, and I need to amplify it with taq
>> polymerase (because that's what I have, and I'm too poor to afford better
>> right now). It's the length that's the main problem, although challenges
>> relating to the specifics of how to amplify it are also troubling me.
>> 
>> Specifically, I need to amplify a ~3.6kb plasmid *from another cloning
>> vector* as a linear molecule such that either amplicon end is complementary*
>> to the other, because this will enhance B.subtilis' ability to take up the
>> DNA and recombine it into a ring-shaped plasmid once more..or at least
>> enhance the plasmid's likelihood of copying itself successfully into a
>> ring-shaped plasmid by rolling-circle-replication before it is lost.
>> 
>> Taq seldom amplifies longer than 2kb, but there are voodoo ways to make it
>> do so. Among these voodoo methods are alternate buffers (apparently removing
>> potassium salt can improve matters?), different cycling conditions, and
(Continue reading)

Permalink | Reply | 0 comments |
headers
DK | 8 Jul 2011 21:16

Re: Long-amplicons and Taq

In article
<740568f1-764d-4381-aa90-15b203e7998b@...>,
chovek69 <ivanoov@...> wrote:

>I regularly amplify >3kb with regular Taq with (NH4)2SO4 buffer (see
>link below) + 1M Betaine and 5min extension step at 65C.
>
>http://www.whitelabs.org/Lab%20Protocols/PCR%20Protocols/kyle.htm

Just curious: why 65C? 5 min total extension for 3 kbp or per every kbp? 

Thanks, 

DK
Permalink | Reply | 3 comments |

Gmane