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Freiberger's Content

There have been 9 items by Freiberger (Search limited from 19-February 19)


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#12729 ABI7000 + SYBR GREEN I

Posted by Freiberger on 22 March 2005 - 08:44 AM in Molecular Biology

Dear Yongyk,

Thanks very much for your very helpful advise! As for the internal standard preparation,
I run across different papers suggesting either 16S rRNA or in vitro transcribtion.

Did you make any experiences with 16S rRNA as an internal standard? It seems for me more obvious to use RNA as a standard since I am looking at the mRNA level in the cells.

Thanks again!
Freiberger



#12591 XmaI puzzle

Posted by Freiberger on 18 March 2005 - 08:30 AM in Molecular Biology

Hi,
I can't imagine that you can do "ligation" with a enzyme buffer - you do need a ligase to do such things ;-).

Have you tried to change the enzyme buffer? Maybe the buffer is contaminated.

Freiberger



#12548 real-time PCR/ABI Prism 7700

Posted by Freiberger on 17 March 2005 - 05:49 PM in Molecular Biology

Hi,

I think you can get an update for the software that comes with the SDS from ABI.

Maybe the update will solve your problem...


Good luck!
Freiberger



#12547 XmaI puzzle

Posted by Freiberger on 17 March 2005 - 05:34 PM in Molecular Biology

Hi!

Do you gel excise + purify your PCR product or do you just run some ul to verify your product?

If you don't gel excise your product I could imagine that you still have some of your PCR template in the mix. What was your template DNA? Does your template might have a XmaI site?

Have you tried to cut w/ SmaI? It's an isochizomere generating blunt ends (could make your subsequently cloning step more difficult).

Good luck!



#12463 RT- PCR?

Posted by Freiberger on 16 March 2005 - 07:19 PM in Molecular Biology

Hi,

Check this out: www.gene-quantification.com

Freiberger



#12262 ABI7000 + SYBR GREEN I

Posted by Freiberger on 14 March 2005 - 04:23 PM in Molecular Biology

Hi everyone!

I am looking for some good advise... I want to do quantitative PCR in order to determine the expression level of some prokaroytic genes using ABI7000 and SYBR GREEN. When I started reading papers about the whole topic I got a little bit scared since there seem to be so many sources of potential errors (standars, primer dimer, standard curve, dilutions.... .

Does anyone can recommend a good tutorial about the ABI7000 or could provide me with a protocol?

Thanks very much for every response!!!!



#11336 cloning from PCR

Posted by Freiberger on 23 February 2005 - 02:05 PM in Molecular Biology

Hi,
The RE Xba I is sensitive to dam methylation. Maybe a reason why you don't see any small fragment is because one of you RE didn't cut...



#11335 Help! E.coli GM48: digestion/DNA degradation

Posted by Freiberger on 23 February 2005 - 01:44 PM in Molecular Biology

Hi,

I got a problem which is going to drive me crazy. In order to use the RE BclI, I transformed my plasmid (pGEM+insert) into E.coli GM48. After extracting the plasmid (NucleoSpin), I set up the digest with BclI as described in the manual. But if I run some of my digest on a gel there is no DNA visible. I run a couple of troubleshooting experiments: it's not the kit, it's not the enzyme, it's not the buffer, it's not the water I use for the digest and I do have the plasmid extracted. Just the combination of buffer+water (no matter what water or buffer I take) degrades my extracted plasmid. 

I also used a different enzyme which is not blocked by dam methylation with the same result!

I would be very grateful if there is anybody who could help me with this issue.
Thank you in advance!

:)

Hi everyone,

I found the source of my problem. In case anybody runs into the same issue, I would recommend to have a closer look at the plasmid extraction manual. In same case there is an additional wash step recommended to remove traces of nuclease activity. That's the key.



#11281 Help! E.coli GM48: digestion/DNA degradation

Posted by Freiberger on 22 February 2005 - 10:44 AM in Molecular Biology

Hi,

I got a problem which is going to drive me crazy. In order to use the RE BclI, I transformed my plasmid (pGEM+insert) into E.coli GM48. After extracting the plasmid (NucleoSpin), I set up the digest with BclI as described in the manual. But if I run some of my digest on a gel there is no DNA visible. I run a couple of troubleshooting experiments: it's not the kit, it's not the enzyme, it's not the buffer, it's not the water I use for the digest and I do have the plasmid extracted. Just the combination of buffer+water (no matter what water or buffer I take) degrades my extracted plasmid.

I also used a different enzyme which is not blocked by dam methylation with the same result!

I would be very grateful if there is anybody who could help me with this issue.
Thank you in advance!

:)




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