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blank and substrate control give OD reads

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blank and substrate control give OD reads

Postby intandiana » Wed Jan 15, 2014 7:21 am

Hi there...I'm new here. Hopefully all of u can solve my problem.

I'm working on screening of xylanase variants to look for the better variant than the wild type in hydrolysis kenaf. the reaction is done in 96 deep well plate by adding substrate (kenaf) + buffer (50mM sodium acetate, pH 5.0) + enzyme solution and incubate for 4 hours in waterbath. after 4hrs incubation, the reaction is centrifuged to collect only reaction solution (without the substrate) then stop solution with DNS and incubate at 100oC, 5min. each experiment is controlled by blank (buffer only), substrate (buffer + substrate) and wild type xylanase.

the problem is the blank and substrate control give higher OD reads than the wild type and variants. I added antimicrobial (tetracyline-HCL) into the reaction mixture but not work.

any suggesstion to help me?? :(
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Re: blank and substrate control give OD reads

Postby jonmoulton » Thu Jan 16, 2014 3:23 pm

Is there anything in the buffer that absorbs light at the wavelength you are using for your OD readings? It might be worth running the buffer in a spectrophotometer and comparing against water at wavelengths near your OD measurement.
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Re: blank and substrate control give OD reads

Postby jonmoulton » Thu Jan 16, 2014 9:46 pm

Here's another approach. Set up a pair of tubes identically, containing everything but the xylanase. Check their OD. Add xylanase to one of the tubes and mix. Check the OD again. That way you control for other potential errors, such as a difference in buffer concentration or a variation in tube thickness (I have no idea what the error is, but starting with identical preps and measuring them before adding enzyme should help you determine that).
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Re: blank and substrate control give OD reads

Postby JackBean » Fri Jan 17, 2014 10:05 am

You centrifuge the reactions in the 96-well plate and then in that you measure absorbance? How can you do that if the bottom is blocked by your substrate?

Can you describe your reactions and controls in more detail including some example absorbances so we can make better idea what you have in there?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: blank and substrate control give OD reads

Postby intandiana » Sat Jan 18, 2014 8:39 am

jonmoulton wrote:Is there anything in the buffer that absorbs light at the wavelength you are using for your OD readings? It might be worth running the buffer in a spectrophotometer and comparing against water at wavelengths near your OD measurement.


Dear jonmoulton,

thanks for your reply.
>>>my buffer is sodium acetate pH 5.0. I'll compare it against water.
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Postby JackBean » Sat Jan 18, 2014 2:37 pm

it doesn't have to be only the buffer but any additive you're using
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: blank and substrate control give OD reads

Postby intandiana » Sun Jan 19, 2014 5:45 am

jonmoulton wrote:Here's another approach. Set up a pair of tubes identically, containing everything but the xylanase. Check their OD. Add xylanase to one of the tubes and mix. Check the OD again. That way you control for other potential errors, such as a difference in buffer concentration or a variation in tube thickness (I have no idea what the error is, but starting with identical preps and measuring them before adding enzyme should help you determine that).


ok good idea. will try it soon.
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Re: blank and substrate control give OD reads

Postby intandiana » Sun Jan 19, 2014 5:57 am

JackBean wrote:You centrifuge the reactions in the 96-well plate and then in that you measure absorbance? How can you do that if the bottom is blocked by your substrate?

Can you describe your reactions and controls in more detail including some example absorbances so we can make better idea what you have in there?


detail method as below;
1) grow inoculum of xylanase variants in 96 multiwell plate, overnight, 37oC
2) expression in deep well plate for 24hrs, 30oC by 5% inoculation
3) centrifuge to harvest the enzyme
4) distribute substrate (pre-treated kenaf) equally to 96 deep well plate
5) add 250ul enzyme (from step 3) + 250ul sodium acetate into the substrate
6) incubate _at_ 50oC, 4hrs in waterbath
7) centrifuge to sediment the substrate
8) pipet 100ul reaction solution and transfer to new 96 deep well plate
9) add 100ul DNS solution, incubate _at_ 100oC, 5min
10) pipet 100ul and transfer into multiwell plate to read _at_ OD540
>>> each experiment is done with blank (buffer sodium acetate only), substrate control (substrate + buffer) and wild type xylanase (with substrate and buffer)
>>>example result from 1 of experiment by OD 540 measurement: blank-0.119, substrate control-0.127, wild type-0.095

hope you understand and can trace down any mistake..
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Re:

Postby intandiana » Sun Jan 19, 2014 5:59 am

JackBean wrote:it doesn't have to be only the buffer but any additive you're using


other additive that I used is antimicrobial reagent-tetracycline HCL which is I used for prevent any contamination in the reaction but I think there is no difference after added this reagent, means that OD blank and substrate control still higher.
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Postby JackBean » Mon Jan 20, 2014 10:55 am

I'd check each component of the reaction mix separately for absorbance against water.

BTW shouldn't your blank have absorbance 0?
http://www.biolib.cz/en/main/

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