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yongyk's Content

There have been 24 items by yongyk (Search limited from 06-August 19)


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#13246 DNA copy number-QPCR

Posted by yongyk on 30 March 2005 - 12:02 PM in Molecular Biology

Dear molcyt,

Are you working with gene expression?


Regards
yong



#13129 Protein Homogenization

Posted by yongyk on 29 March 2005 - 02:35 AM in Protein and Proteomics

Dear friend,

Again, this is regarding to my cloning work. I clone my protein in pQE-30 vector and tranfect into E.Coli (Qiagen product). After induced by IPTG, the bacteria was harvested and protein of interest was purified using Ni-NTA method acroding to manufacturer instruction.

However when I run SDS-PAGE, I still can see a lot of my protein in the cell lysate. My protein is a soluble protein, why it can be mixed with cell lysate??

From MERCK website I got this information that: "soluble protein has to be span at 10,000g at least 30 min before it can be seperated from insoluble protein/homogenized in supernatant."
Is that true?

I will be glad if someone can comman on this

Thank everybody : )

Yong



#13125 cDNA cloning by PCR

Posted by yongyk on 29 March 2005 - 02:18 AM in Molecular Biology

Dear nousheen,

To get the primer for other flanking region of the gene, I think is good to use bioinformatic software and make use of swissprot protein database.

Now you know 15aa (=45bp). load your know 45bp on NCBI and BLAST. From the BLAST search, the programe will show you a lot of similar protein. You can make use the information from the data base predict your reverse primer.

Good luck

Best regards
yong



#13120 How to dissolve over-dried DNA?

Posted by yongyk on 29 March 2005 - 01:42 AM in Molecular Biology

Dear Felix,

I guess you dissolved your plasmid pellet in ddH2O, right?
Try to dissolve in 8mM NaOH.

Next time when you extract any of your DNA, never never let it over dry.
After you discard the supernatant (70% ethanol) try to pipet out the what ever solution remain at the bottum of the tube and let it air dry brifly. Or you can put on thermal cycler block, set at 50-60oC for 2 min (I always do so).

Regards
Yong



#13067 Ni-NTA problem

Posted by yongyk on 27 March 2005 - 06:57 PM in Protein and Proteomics

Dear friends,
Since I purify my protein in denature condition, will it still form agregate?
If it do form agregate, how to get rid of it?

Regards
Yong



#13035 Sequencing 40 kb long vibrio dna

Posted by yongyk on 26 March 2005 - 02:13 AM in Molecular Biology

Dear Sequencer,

I think you gene fragment is simply too large. It might contain a lot of repetative sequence.

regards
yongyk



#13034 gels used for RNA detection

Posted by yongyk on 26 March 2005 - 02:00 AM in Molecular Biology

Dear Vetticus,

Which gel to use is really depend to you RNA fragment size.
I was using acrylamide gel to purify my 46bp RNA probe. I follow protocol privide by Ambion Maxi Script kit. However, the method provided is using radioctive isotop detection.

Regards
yongyk



#13033 PCR help

Posted by yongyk on 26 March 2005 - 01:50 AM in Molecular Biology

Dear kroger,

What MoleculeMan say is very true. What extraction method are you using?
Try to chech ratio of A260:A280.

Good Luck
yongyk



#13032 Agarose or acrylamide gel for DNA?

Posted by yongyk on 26 March 2005 - 01:42 AM in Molecular Biology

Dear monolithis,

Both agarose and acrylamite can be use to seperate DNA. Generally, agarose gel is much more cheeper and esier to prepare compare to acrilamite gel. However, the resolution of agarose gel is no way to compare to acrylamite.
Which one should use is really depend on your purpose.

As I am doing rutine diagnosis work, allI need to know is positive or negative. Thus agarose gel surve the purpose.

I f you doing DNA profilling work like fringerprinting, SSCP which require high resolution, acrylamite gel is the choice.

Normally,gel % of agarose gel is 1.5-3%. Acrylamite gel is about 12%.

Regards
yongyk



#13031 3 bands on RT-PCR

Posted by yongyk on 26 March 2005 - 01:30 AM in Molecular Biology

Dear georgepatra,

There are two posibilities that can make you RT-PCT generate more than 1 band.

First, your primer not specific enough. Not so much during PCR but more important during your RT step. As we know, RT step has to be carry out at lower temperature (~50oC). This might be how the unspecific band come about.

Secondly, some of your RNA had degraded into smaller fragment. Thus the small fragment actually serve a "primer" during PCR. So now you got "extra primer" in your solution. That why you can get multiple band.

Good Luck

yongyk



#12791 tRNA and Real time RT-PCR

Posted by yongyk on 23 March 2005 - 01:05 AM in Molecular Biology

Dear molcyt,
First of all, why your gene has low expression? and why your assay can from primer dimer? (Is your assay not optimized or your primer can form primer dimer?)

I don't know anything about tRNA but can give your a suggestion to get rid of primer dimer.

First when you run melting curve analysis, you should see two peaks. The one with higher Tm is your specific product; the lower Tm is primer dimer.
Let say your melting temperature of the specific product is 85oC and you drimer dimer is 75oC. You can perform your PCR in as following condition
95oC 30s
550C 30s
72oC 30s
80oC 10s*

* set as optical data collection step.

If you collect your optical data at the temperature higher than you primer dimer formation temperature, what ever data collected well be free from primer dimer interference.

so you still can collect a valid data olthough the gene has low expression.

Hope this will work for you. I tried and it work for me.

Best Regards
Yongyk



#12780 ABI7000 + SYBR GREEN I

Posted by yongyk on 22 March 2005 - 09:27 PM in Molecular Biology

Dear Freiberger,

Yes, you are right, since you are quantifying gene expression level, the best way of doing is measuring the expression of housekeeping gene, so that you can normalized the signal of both genes.

All the while I have been doing absolute quantification, but not relative quantification (using internal control). Sorry I can't give instraction in detail, i am sorry about that.

However, when you using SYBR Green, is hard to differentiate two PCR product by melting curve analysis, unless both of the GC% of your both genes are distinct enough.

Regards
Yong



#12707 Ni-NTA problem

Posted by yongyk on 21 March 2005 - 09:54 PM in Protein and Proteomics

hi
i got this problem once and that was a problem of column equilibration...

<{POST_SNAPBACK}>


Hi,
Could you be more specific?

Thank

Yongyk



#12706 Express a vaccine protein

Posted by yongyk on 21 March 2005 - 09:49 PM in Molecular Biology

Dear rdrun,

Please talk to these person: Prof. Dr. Thong Kwai Lin of University Malaya, Malaysia.
She is S. typhi expert in my country.

her email: thongkl@umcsd.um.edu.my

Good luck
Yongyk



#12705 Help:how much gDNA is needed?

Posted by yongyk on 21 March 2005 - 09:39 PM in Molecular Biology

dear flyinghorse,

It really depent on what vector are you using. for me, I use 100ug before and it work.

Good luck
Yongyk



#12704 ABI7000 + SYBR GREEN I

Posted by yongyk on 21 March 2005 - 09:27 PM in Molecular Biology

Dear Freiberger,

Since you are trying to determine expression level of certain gene, you have to do a real-time quantitative RT-PCR.

First you have to prepare your standard in RNA form. I would recomemd you to use in vitro-transcription methods rather than cloning.

Go to get an Ambion MAXI script in vitro transcription kit and follow the protocol provided, you will able to get some high abundant and good quality of RNA.

Purify your RNA. Do not use any commercial kit if your fragment is around 100-200bp. Because as the fragment is too small, it woun't stuck on the membrane of the spin column when you spin it. Try to follow conventional method. More useful.

Quantify your RNA using spectrophotometer and convert them into copy number, you should able to get around X e11 copy of RNA.

Aliquot you stndard stock and store in -20. Newly prepare a 10 fold serial
dilution each DAY when you run your assay. The remainder put into +4 and you can use within the same day.

I am using BioRad iCycler, so can not give you any info about ABI prism.

I hope it work for you

Best Regards
Yongyk



#12645 ELISpot for IFN-g

Posted by yongyk on 21 March 2005 - 01:00 AM in Immunology

I am looking for a protocol which can work for ELISpot to detect interferon-gamma. I will inject the antigens to the mice and trying to observe the cellular response of the mice after 2 weeks.

Thank you so much.


Yong :(



#12533 DNaseI have affect with RT-PCR or not

Posted by yongyk on 17 March 2005 - 12:30 PM in Molecular Biology

Dear penguin_th

I have few question to ask:
1) since you are using RNA extraction kit, why you were doing PCR? Do you means you run RT-PCR first, don see band and followed by second PCR?

2) Have you get this set of primer work before?

Best Regards
yongyk



#12532 real time qPCR

Posted by yongyk on 17 March 2005 - 12:20 PM in Molecular Biology

Dear Ribosoul,

Let me clearify with you. What do you means by "more copies". Are you reffering to initail copies or DNA amount of your PCR product?

1) Ct Value tells you the information of the initial copies.

2) RFU during the amplification curve will tell you the amount of your total PCR product (specific product + primer dimer).

3) RFU during the melting curve will tell you amount of specific PCR product.

Since standard curve is build base on Ct value (initial copy) thus there is no way to tell the exact number of the end product. That why I said is "qualitative".

You can only only say a pcr product with a higher piek has more copies (end product) when both of your PCR product are in similar size. Why?

Just compare a fragment of 350bp and a fragment of 105bp. The larger one will incorperated more SYBR Green molecule than the smaller one, and end up with higher RFU.

When you build a standard curve by performing 10 fold series dilution, you will see your height of you melting peak decreesing from the high to low concentration.
when you take the PCR product and run a gel, you will see the sample with highest melting peak will have nost intense band, whereas the lowest peak you might not be able to view on gel.

Thank you

Best Regards
yongyk



#12530 cDNA cloning by PCR

Posted by yongyk on 17 March 2005 - 11:44 AM in Molecular Biology

dear nousheen,
Can I know from which organism that you isolated your protein?
How many base pair that you know?

I need to know those information before I can know what to do.
Thank.

Regards
yongyk



#12476 Ni-NTA problem

Posted by yongyk on 17 March 2005 - 12:47 AM in Protein and Proteomics

Dear friends,
I am dealling with bacteria OMP. I had cloned the OMP gene into QiaExpress vector pQE30 and was N-terminal tagged with His. However, when I purify my protein, I can't get my protein in elution buffer, but they all present in washing buffer. What is happening?

Thank You

Regards
Yongyk



#12469 PCR help

Posted by yongyk on 16 March 2005 - 08:17 PM in Molecular Biology

Hi,
Do you quantitate your template prio PCR? When yoy have smeared band from top to the bottom of the gel most likely due to the Excessive omount of templete.

When you carry out DNA quantification be aware of you DNA purity. The ratio of A260:A280 has to be > 1.5 and you anly put < 1ug per 50ul reaction.

Try it out! Good luck!

Yongyk :P



#12468 real-time multiplex PCR.

Posted by yongyk on 16 March 2005 - 08:00 PM in Molecular Biology

Dear friends,
I wish some body can tell me what going on.
I am runing real-time multiplex pcr for subtyping for yeast. I have three probes FAM. Texas Red and Cy5 and there share both forward and reverse primer.
When I run the assay, FAm got very high Fluorescence unit (around 3000), but my Texas Red and Cy5 is like not even 150. How ever their Ct value are around 13-15 cycle.

What determine the Fluorescence unit for real-time assay?

Regards
Yong



#12467 real time qPCR

Posted by yongyk on 16 March 2005 - 07:49 PM in Molecular Biology

Dear Ribosoul,

The height of the piek represent Relative Fluorescence unit (RFU). It tells you qualitatively the amount of the specefic product contain in your tube.

I think the more relevent info getting from the dissasosiation curve is the specific meltting temperature. Dont't worry about the height.

Regards
Yongyk :P




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