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jerryshelly1's Content

There have been 197 items by jerryshelly1 (Search limited from 05-April 19)



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#162446 DNA extraction impurities

Posted by jerryshelly1 on 14 November 2013 - 07:20 AM in Molecular Biology

PM Sayk and see if he/she worked out the problem.




#162119 DNA extraction impurities

Posted by jerryshelly1 on 05 November 2013 - 10:31 AM in Molecular Biology

It may affect your DNA concentration, but not the residual protein. If they are immune compromised (i.e. low WBC's) you would expect a lower than average DNA concentration, but the overall consistency of the blood would be similar.

 

As far as the chemotherapy, I do not have enough knowledge on that to make a guess. Are you only seeing this phenomenon in you cancer and chemo patients? Do your healthy controls show this?

 

I checked the wizard protocol and it is very straight forward. Do you see any clumps in the whole blood before you begin your extraction? Is this a recent problem that was absent in the past?




#162114 DNA extraction impurities

Posted by jerryshelly1 on 05 November 2013 - 08:48 AM in Molecular Biology

It was a silly question, but I am just making sure the blood was homogenous before you began your prep. Blood will separate into its respective components fairly quickly and I have seen people take the plasma or the RBC's while leaving the buffy coat almost undisturbed.




#162520 DNA extraction impurities

Posted by jerryshelly1 on 16 November 2013 - 05:15 PM in Molecular Biology

What kit are you using? Make sure all your solutions are homogenous. If you continue to have problems, you can contact the manufacturer and see if there have been additional problems with your particular lot number. If anything, they may give you a new kit. I'm not certain though.




#162536 DNA extraction impurities

Posted by jerryshelly1 on 17 November 2013 - 09:57 AM in Molecular Biology

It is usually standard, but look and see what anti-coagulant is used on the tube (heparin, etc...)




#162558 DNA extraction impurities

Posted by jerryshelly1 on 18 November 2013 - 07:01 AM in Molecular Biology

It may be worth it to contact another company and see if you can try one of there kits for free. Since you regularly isolate blood, it will be beneficial for them. I have had good success with EZ Bioresearch's MW blood isolation kit (competitively priced).

 

It makes me worried that two people have commented on the inefficiency of this kit.




#162556 DNA extraction impurities

Posted by jerryshelly1 on 18 November 2013 - 06:18 AM in Molecular Biology

I honestly don't know. EDTA is a good anticoagulant. Maybe try contacting Promega and see if they have any advice.




#162080 DNA extraction impurities

Posted by jerryshelly1 on 04 November 2013 - 08:28 AM in Molecular Biology

Are you using fresh blood and/or are you properly resuspending it before beginning the isolation? If it is older blood, it may contain fibrin which can induce clotting and will make it more difficult to adequately remove all protein.




#162123 DNA extraction impurities

Posted by jerryshelly1 on 05 November 2013 - 11:44 AM in Molecular Biology

The only thing I can suggest is taking some "junk" blood and running a couple of different conditions side by side.

 

Maybe...

Increase centrifugation time on a sample, precipitate protein at 4C for 10min?, increase amount of precipitation solution?




#162031 DNA extraction impurities

Posted by jerryshelly1 on 02 November 2013 - 01:50 PM in Molecular Biology

If you are extracting blood, it looks like your protein precipitation buffer is no longer viable. The red color is due to the protein hemoglobin being present in the solution. I would double check your protocol and make sure that you are allowing for proper precipitation of your protein and also check to see if your precipitation buffer is coming out of solution.

 

Are you centrifuging your samples adequately each time? Is it possible you are overloading the kit with blood each time?

 

As far as the brown pellet, it looks like you still have cellular debris in your sample.




#167134 I have a careless boss

Posted by jerryshelly1 on 18 April 2014 - 06:19 AM in Venting and Counseling

Sorry to hear that. It is extremely unfortunate that you are in this position. How can this individual maintain her funding if she is an overall terrible mentor? It seems that the administration would look poorly on her overall negligence.




#161626 Runing problems

Posted by jerryshelly1 on 24 October 2013 - 08:46 AM in SDS-PAGE and Western Blotting

Hey Vemovied,

 

I don't see a pic.  Can you try to attach it again?




#166862 crystallisation of PBS (10X)

Posted by jerryshelly1 on 09 April 2014 - 11:55 AM in General Lab Techniques

We use those and they work fine. Just practice better aseptic techniques. If it is common lab reagent, make sure everyone is informed on the proper handling of the chemicals. Sounds like someone was careless pouring the solution and introduced a contaminant.




#164837 crystallisation of PBS (10X)

Posted by jerryshelly1 on 05 February 2014 - 09:12 AM in General Lab Techniques

Do you always use that much disodium phosphate? We only use ~14g, it is possible that you oversaturated the solution and it is precipitating out. 




#165302 Gel Electrophoresis: Help needed

Posted by jerryshelly1 on 21 February 2014 - 09:40 AM in Molecular Biology

Has this protocol worked before? Homogenization wouldn't be enough to rupture the cells for DNA extraction. Adding sodium acetate will do nothing for DNA precipitation if the DNA is still contained within the membrane. I would add a lysis buffer to your cells and nix the vigorous homogenization.

 

Edit - Hobglobin beat me to it. Listen to him.




#161316 how to remove affinity tag completely

Posted by jerryshelly1 on 16 October 2013 - 10:39 AM in Protein Expression and Purification

Can you do an immunoprecipitation for your tag? Should leave all untagged protein in your lysate.




#161642 midi-prep

Posted by jerryshelly1 on 24 October 2013 - 12:11 PM in General Lab Techniques

Is this a kit based extraction or a crude phenol-chloroform extraction? 

 

When DNA concentrations are low, you can precipitate your DNA out with 10% NaOAc/EtOH for 30min to O/N at -80C. You also add glycogen if your ethanol solution if your DNA is being difficult.

 

Edit: Oh the typos...




#166876 PCR not working

Posted by jerryshelly1 on 09 April 2014 - 01:30 PM in PCR, RT-PCR and Real-Time PCR

Silly question, but you are trying to amplify each exon independently correct... Running your FWD/REV primer pairs through NEB and IDT gives a two Tm's that are relatively close. Make sure that your primers and all of your reagents are diluted properly before running and give gradient PCR a try.




#166863 PCR not working

Posted by jerryshelly1 on 09 April 2014 - 12:01 PM in PCR, RT-PCR and Real-Time PCR

It sounds like the annealing temperatures of your primers are way to far off. Does the sequence of ABCD1 make it impossible to design primers that have similiar annealing temps? Are you able to post your primer sequences?




#166868 PCR not working

Posted by jerryshelly1 on 09 April 2014 - 12:25 PM in PCR, RT-PCR and Real-Time PCR

What exactly are you trying to do? Are you setting up a standard PCR? You could trying running a gradient PCR or add betaine or DMSO to your reactions.




#161100 Colony PCR positive and Digestion negative?????

Posted by jerryshelly1 on 10 October 2013 - 07:41 AM in Molecular Cloning

Yes, your control sounds good. Your CIP treated vector with ligase should not religate unless your phosphatase is not working properly; especially with two non-compatible cohesive ends.

 

How close together are your two restriction sites? 3, 3, 12 bp?

 

Did you mean you have had success with colony PCR using these primers in the past?

 

You have a very interesting problem...




#159695 Autoclaving water to go into the incubator

Posted by jerryshelly1 on 30 August 2013 - 09:06 AM in Tissue and Cell Culture

 

Same here. We autoclave regular tap water and supplement the water with SDS. Distilled and Milli-Q water will rust your incubator.

 

What is the purpose of adding SDS? As an anti-bacterial?

 

We have seen it reduce the growth of bacteria. It is especially helpful if someone accidently splashes media into the water tray.




#161361 Colony PCR positive and Digestion negative?????

Posted by jerryshelly1 on 17 October 2013 - 10:09 AM in Molecular Cloning

The confirmed colonies you have were checked by the same REs prior to sequencing?




#161105 Colony PCR positive and Digestion negative?????

Posted by jerryshelly1 on 10 October 2013 - 08:54 AM in Molecular Cloning

How far apart are your RE sites on your vector before digest?




#161097 Colony PCR positive and Digestion negative?????

Posted by jerryshelly1 on 10 October 2013 - 07:12 AM in Molecular Cloning

There was a paper published a number of years ago showing colony PCR's susceptibility for a high number of false-positives.  The authors suggested that the false positives may be the result of the ligated vector + insert's inability to enter the bacterial cell. The correct ligation product would therefore be in your mixture of cells and may "stick" to the outside of your bacterial cell via a LPS. Who knows if this is the correct case, but I know I have had very little success with correctly identifying constructs via colony PCR. I usually avoid this procedure.

 

In your controls, do you mean that your vector only did not have ligase? It sounded like you just had ligation of your originally cut vector; however, it doesn't make any sense why your vector only would have fewer colonies...





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