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jerryshelly1's Content

There have been 197 items by jerryshelly1 (Search limited from 01-June 19)



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#160616 Weighing harvested cells

Posted by jerryshelly1 on 24 September 2013 - 01:26 PM in Tissue and Cell Culture

Figure out your OD. You could easily calculate "mg" of cells from this number.




#161648 question about sequential transfection several plasmids in 293T and COS7 cells

Posted by jerryshelly1 on 24 October 2013 - 12:45 PM in Tissue and Cell Culture

How are you transfecting your cells? Are you doing a transient transfection, waiting two days and then transfecting again?

 

Do you linearize your plasmids when you transfect your cell with multiple plasmids. If not, I would at least linearize the LacO plasmid. That guy is huge and the cell may have some difficulty taking it up.

 

I would start by transfecting your lowest transfection efficiency plasmid, selecting for it and then proceeding with the others. How are you selecting for your plasmids? Does each plasmid harbor a different mammalian resistance or are you doing downstream assays to check the expression of each plasmid?




#161674 SNP in the promoter region

Posted by jerryshelly1 on 25 October 2013 - 06:49 AM in Molecular Cloning

If it is a known SNP you can go to Ensembl and see what is known about it. Another good resource is dbSNP.




#166162 Cloning pDsRed-Monomer-N1

Posted by jerryshelly1 on 19 March 2014 - 11:15 AM in Molecular Cloning

It would be better if you could provide a detailed description of what you are doing. There are tons of cloning resources on this website that you might consider browsing through. My most common cause of ligation failure is proper digestion of my PCR fragments. You usually get good digestion with a vector due to its size and shape, but PCR fragments can be problematic depending on the size.

 

I usually like to test the efficiency of my ligation by running a simple PCR with a primers that recognize both the vector and PCR fragment. Amplification is a good indication that some ligated products are in your sample.

 

What are your JM109 cell controls like? Do you observe a good transformation efficiency?




#162453 Freshly prepared RPMI plus SERUM= Colour change???

Posted by jerryshelly1 on 14 November 2013 - 09:14 AM in Tissue and Cell Culture

You just shocked the system. When I add antibiotic to my tissue culture media (DMEM), I see an immediate color change. After letting the media sit for ~12hrs, the color is restored. You just need to keep in mind that pH indicators are present in the media and when a solution is added, it needs to fully equilibrate. I wouldn't worry as long as you followed the protocol.

 

Try gently mixing the solution or placing it in a water bath for about 30mins.




#165296 can buffer ever go bad?

Posted by jerryshelly1 on 21 February 2014 - 09:18 AM in Tissue and Cell Culture

Aside from contamination and precipitation, you should be fine.




#163896 suicide muatagenesis

Posted by jerryshelly1 on 07 January 2014 - 09:38 AM in Molecular Biology

Phage gives a good answer here:

 

http://www.protocol-...posts/4079.html




#164378 cloning pEGFP-N1

Posted by jerryshelly1 on 21 January 2014 - 08:37 AM in Molecular Cloning

Here is a plethora of information for you to sort through... http://www.protocol-...ecular_Cloning/




#166424 Deleting 7Mbp chromosomal region

Posted by jerryshelly1 on 29 March 2014 - 11:42 AM in Molecular Cloning

Do you need to delete it completely or can you just disrupt the sequence to hinder production?




#160391 When cell media becomes "used up", it acidifies

Posted by jerryshelly1 on 19 September 2013 - 06:26 AM in Tissue and Cell Culture

Go review Glycolysis and the Krebs cycle and look for any enzyme with the work "dehyrogenase" in it.  This will show you where the protons are being generated. I am not sure if I understand the second question. My only knowledge on that would be that protons are released into the "actual media" due to symport and antiport for the use of bringing in the nutrients that are present in your DMEM (or other media). This is the energy that is required for specific uptake of some of your nutrients.

 

This is as much as I can elaborate without reviewing the relevant literature.

 

Hope this helps.




#160370 When cell media becomes "used up", it acidifies

Posted by jerryshelly1 on 18 September 2013 - 12:07 PM in Tissue and Cell Culture

As with any living tissue, cellular respiration (glycolysis and oxidative metabolism) generates protons that can reduce the pH of cell media. It is the same as a respiring tissue within a living organism; however, a circulatory system is absent to replenish the media.

 

*On a side note. Pour bleach into your media and this will demonstrate that H+ ions are responsible for your pH shift. I do not know why I get such a thrill by doing that.




#162395 What stage of growth are the cells I counted?

Posted by jerryshelly1 on 13 November 2013 - 07:00 AM in Tissue and Cell Culture

Do you have multiple cell counts at different time points or are you just given one number? Go to ATCC and find the growth curve of your cells and from here you can determine what phase your cells were in.

 

ATCC:

http://physics.cance...-29_SOP-508.pdf




#166161 Purified DNA fragments are bigger than unpurified

Posted by jerryshelly1 on 19 March 2014 - 11:10 AM in Molecular Biology

Double check your cut site to make sure that you are getting the correct dropout size. Salt concentration can have an effect on the migration of DNA in gels, but from your post I would assume that the size difference is significant.




#160776 cDNA preparation from degraded RNA

Posted by jerryshelly1 on 30 September 2013 - 05:59 AM in Molecular Biology

Do you see the 50bp product in your controls without primer? You have used this primer before with no additional bands or is this a recently designed primer? Does the appearance of the additional band only occur with this sample?




#160916 Calibrator sample has zero expression of target gene.

Posted by jerryshelly1 on 04 October 2013 - 06:56 AM in Molecular Biology

Are you trying to compare the fold increase of your cytokine at time point zero vs. a later time point?

 

There are a couple of different ways you can approach your quantification depending on what you are trying to show.  Does the treatment of your sample suddenly switch on your cytokine or is your cytokine usually differentially expressed? Are you trying to compare your cytokine expression to the expression of a common ubiquitous cytokine?




#162294 no protein expression

Posted by jerryshelly1 on 11 November 2013 - 12:10 PM in Molecular Biology

That doesn't really help. How did you design your primers for insertion into your vector. Can you paste or attach the sequence that you used to engineer your primers for RE digestion and subsequent ligation?




#162288 no protein expression

Posted by jerryshelly1 on 11 November 2013 - 09:51 AM in Molecular Biology

Have you double checked to make sure you cloned everything in the correct frame?




#162413 Increasing specificity of allele-specific Tq probes?

Posted by jerryshelly1 on 13 November 2013 - 12:25 PM in PCR, RT-PCR and Real-Time PCR

I am really confused. You designed this to test whether your deletion was present in a large portion of cells. You are looking at relative fluorescence to see how many cells have the deletion, as compared to a non-deletion containing control? What you see is that your "deletion containing cells" do not have the relative fluorescence that you were hoping for. So, you are looking for an easier method to determine the ratio of cells that contain the deletion?




#160777 excess rna sample affect qpcr?

Posted by jerryshelly1 on 30 September 2013 - 06:01 AM in PCR, RT-PCR and Real-Time PCR

I usually use 2ug of RNA and get good, consistent results.  The amount of RNA will not affect the qPCR, because your cDNA protocol should require the degradation of your RNA products following Reverse Transcription.

 

Edit: The ending amount of RNA will not affect your qPCR data.




#161686 GADPH does not express

Posted by jerryshelly1 on 25 October 2013 - 12:44 PM in PCR, RT-PCR and Real-Time PCR

Are any other genes being amplified from your ovarian samples? It is possible that your samples have been degraded. Try your primers with freshly extracted/prepared DNA.




#160197 T7 and M13 primers two band amplification

Posted by jerryshelly1 on 12 September 2013 - 09:18 AM in Molecular Cloning

Did you fragment have two unique RE sites? Maybe you have a double insert of your fragment.  What are the sizes of the two bands and what is the size of your fragment? How far away is the T7 and M13 from your insert?




#160893 Protein extracted from RNAlater tissues is blue... Help!

Posted by jerryshelly1 on 03 October 2013 - 02:10 PM in Protein and Proteomics

Is RNAlater a detergent?




#166427 Electrophoresis samples and volumes

Posted by jerryshelly1 on 29 March 2014 - 11:54 AM in Molecular Cloning

1) I never add water. Are you adding water to get your DNA dye down to 1X? When I set up my PCR I add a consistent concentration of DNA so I can quantify my amplicons using the DNA ladder.

 

2) You probably don't add water here because you would overload the well and your sample mix would spill out. 

 

3) Use one lane if the well can accommodate the entire sample. This will reduce the total amount of agarose an reduce the volume of reagents that will be required for gel purification.

 

4) No. Review the methodology behind electrophoresis on this website or wiki.




#162249 Western blotting

Posted by jerryshelly1 on 09 November 2013 - 05:39 PM in Protein and Proteomics

I wouldn't use it.  If you want to check the efficiency of your gel, you can check your membrane post transfer using ponceau stain.




#160198 Protocol for qPCR using the ABI SYBRŪ Green PCR Master Mix

Posted by jerryshelly1 on 12 September 2013 - 09:26 AM in PCR, RT-PCR and Real-Time PCR

I have used SYBR Green.  I usually used a total reaction volume of 12uL. The final primer concentration would have been 2-10uM (I played around with this and the results stayed consistent - trying to determine the minimal amount of primer I could use - check SYBR green protocol). I would add 0.5uL of a 2ug cDNAtemplate. I would add 6uL of the SYBR Green master mix.

 

My thermal cycling program:

Doublets of the reaction mixture were loaded and cycled once at 50ºC (2mins), and 95ºC (10mins); 40 cycles at 95ºC (15secs), 58ºC (30secs), and 72ºC (30secs); and a final cycle at 95ºC (15secs), 60ºC (20secs), 95ºC (15secs), and 60ºC (15secs).

 

The 58C will be unique to your primer Tm's.  I would always try to design your primer to a specific Tm.  This will allow you to perform very diverse RT-PCR's simultaneously (saves plates and optical film).

 

Good Luck.





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