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



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#165295 Why when disigning primers for RTPCR, you need to chose a region where introns a

Posted by jerryshelly1 on 21 February 2014 - 09:15 AM in PCR, RT-PCR and Real-Time PCR

Consider what your RT-PCR primers are recognizing. When you make cDNA, you are generating the transcript from RNA. By having the primers extend across the intron, you are guaranteeing that you are only reading the RNA expression levels and not any DNA that made it through your purification steps. Does this make sense? 




#161774 Why is my insert missing from my plasmid?

Posted by jerryshelly1 on 28 October 2013 - 10:45 AM in Molecular Cloning

We store them for up to 14days. After that, weird things can happen.

 

Has you lab ever considered switching to a crude phenol-chloroform extraction method to save money. This method can be done when trying to isolate ligation products and the midiprep columns can be saved when preping large quantities of the sample.

 

Edit - Storage will probably depend on your E. coli cell line. This is in reference to DH10beta. I haven't tested on additional cell lines.




#161768 Why is my insert missing from my plasmid?

Posted by jerryshelly1 on 28 October 2013 - 09:24 AM in Molecular Cloning

Select colonies and screen by PCR using an insert- and vector-specific primer.




#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.




#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.




#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




#160577 What plasmid sizes do you work with for SDM?

Posted by jerryshelly1 on 23 September 2013 - 10:29 AM in Molecular Cloning

Voter-phobia Anonymous




#161153 What is your favorite method for making chemically competent E.coli cells?

Posted by jerryshelly1 on 11 October 2013 - 10:59 AM in Molecular Biology

I have tried multiple methods.  My favorite is the Hanahan protocol. Our lab never had any rubidium chloride, but I did scrounge some from an adjacent lab.  I tried the Hanahan protocol side-by-side with rubidium chloride, potassium chloride, and cobalt hexamine chloride.  They all produced negligible transformation efficiencies.

 

The biggest factor is the efficiency of the cells you start with, the media you grow your cells in, the temperature at which you prepare your cells, and the method you handle them. This will drastically affect the efficiency of your cells.

 

I used to work in a similar environment.  Why spend $200.00 for 1mL of competent cells when you could easily make 10mL of competent cells for a fraction of the cost. Comparing my cells to the manufacturer indicated very similar efficiencies. I always try to save money wherever possible.

 

Edit - Incoherent sentence




#161224 What is your favorite method for making chemically competent E.coli cells?

Posted by jerryshelly1 on 14 October 2013 - 06:02 AM in Molecular Biology

Sure.  I will post the one I use when I hit my bench today.




#161231 What is your favorite method for making chemically competent E.coli cells?

Posted by jerryshelly1 on 14 October 2013 - 07:46 AM in Molecular Biology

Hey guys,

 

Two pasted documents. The original protocol and the modified one. Hope this helps.

 

First:

 

XL-10 and Sure-2 Competent Cell Preparation:

 

Day 1:

·      Make appropriate buffers and Media.

·      Inoculate 2mL of media with frozen cell stock (Starter Culture).

·      Incubate O/N at 37°C.

·      TB Buffer was used for both.

·      SOB+ Media was used

 

Day 2:

·      Add fresh buffers to media.

·      Inoculate 2mL culture into 250mL of media.

·      Shake/Incubate at 25°C until OD400-500 (Smaller OD provides better efficiency).

·      In cold room, Transfer cells to cold 50mL falcon tubes and incubate on ice for 30min.

·      Centrifuge cells at 4000xg for 10min at 4°C.

·      In cold room, decant the supernatant and keep the tubes inverted for several minutes to drain off excess media.

·      Gently resuspend cells in 20mL (per 250mL of appropriate buffer).

·      Incubate on ice for 20min.

·      Centrifuge cells at 4000xg for 10min at 4°C.

·      In cold room, decant the supernatant and keep the tubes inverted for several minutes to drain off excess media.

·      Gently resuspend cells in 10mL (per 250mL of appropriate buffer).

·      Incubate on ice for 20min.

·      Add 350mL of DMSO (per 250mL of cells), dropwise and mix gently.

·      Incubate on ice for 10min.

·      Aliquot appropriate amount of cells to chilled eppendorf tubes.

·      Shock-freeze cells in ethanol dry ice bath

·      Store cells at -80°C.

 

Growth Kinetics:

2hr

·      Sure2 – 0.030

·      XL-10 – 0.005

3hr

·      Sure2 – 0.097

·      XL-10 – 0.034

4hr

·      Sure2 – 0.221

·      XL-10 – 0.051

5hr

·      Sure2 – 0.341

·      XL-10 – 0.090

6hr

·      Sure2 – 0.458 (Harvested)

·      XL-10 – 0.084

 

*At this point it was obvious the XL-10 would take 16+ hrs to grow.  I switched the cells to 18°C and let them grow O/N (7:20pm – 9:00am).

 

22hr

·      XL-10 – 0.536 (Harvested)

 

 

*I checked the efficiency with BME, 1mM CaCl2 and plain at 12 hr.

·      XL-10: 8.1e7

·      XL-10 BME: 2.0e7

·      XL-10 1mM CaCl2: 9.5e8

·      Sure2: 4.6e8

·      Sure2 BME: 1.1e8

·      Sure2 1mM CaCl2: 9.4e7

 

Second:

 

*For Sure-2 cells use NZY+ media.

Protocol for the preparation of competent cells

Andreas Leibbrandt

Source: modified Hanahan procedure after Methods Enzymol. 1991; 204:63-113

 

Buffers to render cells competent:

  • DH5a ® FSB + 5% sucrose
  • XL10-Gold ® FSB or TB (Will use TB buffer, unless I can find HexCo(III)Cl)
  • TOP10 ® CCMB80

            FSB buffer ± sucrose:            10 mM KOAc pH 7.5

                                                100 mM KCl

                                                45 mM MnCl2

                                                10 mM CaCl2

                                                3 mM HexCo(III)Cl  - Cobalt (III) Hexamine Chloride

                                                10% glycerol

                                                [5% sucrose]

                                                pH adjusted to 6.4

 

            TB buffer:                        10 mM PIPES pH 6.7

                                                55 mM MnCl2

                                                15 mM CaCl2

                                                250 mM KCl (Good Substitue)

 

            CCMB80 buffer:            80 mM CaCl2

                                                20 mM MnCl2

                                                10 mM MgCl2

                                                10 mM KOAc pH 7.0

                                                10% glycerol

                                                pH adjusted to 6.4

Material check list:

500 ml of SOB or SOB+ medium (order from IMP media kitchen; add 6.25 ml of 1M MgCl2 and 6.25 ml of 1M MgSO4 prior to use, i.e. SOB+), use ~250 ml per E. coli strain

o   SOB: for CCMB80-competent cells, i.e. Mach1 and TOP10 cells

o   SOB+: for FSB-competent cells, i.e. DH5a and XL10-Gold cells

o   SOB+, 40 mg/ml Cam, 80 mg/ml Tet: for XL10-Gold cells

2 l Erlenmeyer flask with red lid (ask the IMP media kitchen to rinse and autoclave as for cell culture glassware)

0.5 l Erlenmeyer flask (ask the IMP media kitchen to rinse and autoclave as for cell culture glassware)

10 ml Falcon 2059 tube

pre-cooled CCMB80, TB, or FSB buffers

pre-cooled 1.5 ml Sarstedt screw cap tubes (from IMP store)

pre-cooled Eppendorf Combitip 5 ml

pre-cooled Falcon tubes, 50 ml

pre-cooled serological pipettes (5 and 10 ml)

ART200 tips

Vortexer in the cold room

liquid N2 or EtOH/dry ice bath in the cold room

ice basket(s) to incubate cells and transfer them from the centrifuge to cold-room

 

 

DAY 1-2

·      pick 5 single colonies and resuspend by gentle vortexing in 1.5 ml of SOB(+) in a Falcon 2059 tube

o   e.g. from a 10-6 dilution prepared from a frozen stock of competent cells, plated on SOB(+)  agar plates and grown o/n @ 37°C

o   alternatively, scrape off some cells from a frozen glycerol stock and resuspend by gentle vortexing in 1.5 ml of SOB(+)  in a Flacon 2059 tube

·      inoculate in 20 ml of SOB(+)  in a 0.5 l Erlenmeyer flask and grow @ 18°C until the culture becomes turbid

DAY 3-4

·      on the next day, dilute 1:100 in fresh SOB(+)  medium and grow cells to an OD600 of ~0.35-0.6 @ 18°C

o   growth @18°C is very slow, so it might be best to start of ~noon the day before to finish the preparation on the next day

·      transfer cells to 5 cooled 50 ml Falcon tubes, and incubate on ice for ~15-30'

o   optionally: prepare glycerol stock, i.e. cells 1:1 with 60%SOB, 40% glycerol

·      spin down cells @ 4000 rpm for 15' at 4°C

o   don't forget to pre-cool the centrifuge and centrifuge containers

·      in the cold room, decant the supernatant and keep the tubes inverted for several minutes to drain off excess media

·      pool the cells (i.e. 5 Falcon tubes) by resuspending the cell pellets carefully (by gentle vortexing or pipetting) in 1/80-1/85 of the original volume in the respective buffer (i.e. for 250 ml of cells, use 3 ml of buffer)

·      incubate bacteria on ice for 20'

·      for FSB preparations, add 3.5% DMSO (105 µl DMSO/3 ml buffer/ 250 ml SOB(+)) from a freshly thawed aliquot of DMSO to cells, mix by gently swirling the tube and incubate on ice for 10'

o   DMSO is stored @ -20°C; remove an aliquot at the beginning of the procedure since it takes a while to defrost

o   apply DMSO drop by drop to the center of the solution and gently swirl the mixture

·      add the same volume of DMSO as before, mix, and further incubate on ice for 5'

·      aliquot 0.2 ml of DMSO-treated competent cells into pre-cooled 1.5 ml Eppendorf tubes and shock-freeze competent cells in liquid N2 or an EtOH-dry ice bath, store cells @ -80°C

o   aliquot cells by using the Eppendorf Multipette with a pre-cooled 5 ml Combitip

 

 

NZY+ Broth (per Liter):

 

10 g of NZ amine (casein hydrolysate)

5 g of yeast extract

5 g of NaCl

Add deionized H2O to a final volume of 1 liter.  Adjust to pH 7.5 using NaOH.

Autoclave.

Add the following filer-sterilized supplements prior to use:

12.5 ml of 1 M MgCl2

12.5 ml of 1 M MgSO4

20 ml of 20% (w/v) glucose (or 10 ml of 2 M glucose) 

 

 

 

*RbCl and Hexamine Chloride can be substituted with KCl. The efficiencies do not differ substantially. 




#167742 What brand of gloves do you prefer?

Posted by jerryshelly1 on 12 May 2014 - 07:44 AM in General Lab Techniques

I use microflex when handling more infectious materials and I use distinct gloves when performing non hazardous work. Make sure you are using the gloves before the printed expiration date. If the webbing keeps ripping, consider using the next biggest glove size.




#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.




#163334 Weirdest thing with DNA dye and agarose gel

Posted by jerryshelly1 on 11 December 2013 - 02:46 PM in General Lab Techniques

Did someone photobleach the safe stain?




#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.




#166953 website for primer design

Posted by jerryshelly1 on 11 April 2014 - 05:51 AM in PCR, RT-PCR and Real-Time PCR

DNASTAR has excellent genomic software and I believe the do offer free trials. A great data mining website is highwire.standford.com. I prefer to use this website instead of Google when performing a search using keywords. I found this paper via Highwire and it looks like primers for exons 1-6 are listed.

 

http://www.plosone.o...resentation=PDF

 

Good luck!




#165306 WBC count in rabbit's synovial fluid

Posted by jerryshelly1 on 21 February 2014 - 10:25 AM in Animal and Zoology

I'm going to go through all the articles, but take a look at these highwire results.

 

http://highwire.stan...etype=1&x=0&y=0




#162144 UV analysis of gel

Posted by jerryshelly1 on 06 November 2013 - 06:27 AM in -Molecular Biology-

People in our lab do if for easy cleanup. I have also seen people place a small piece of saran wrap on the illuminator before placing their gel down.

 

I think it is a waste of time, but if you are using a common lab fotodyne (for agarose, acrylamide, blots, etc.) it may be a good idea to place something down. I personally don't. I just wipe the glass down throughly.

 

Edit - It is also a pain in the ass to get a decent picture without bubbles when there is another material separating your gel from the glass.




#161801 Tool for mapping references to endnote style?

Posted by jerryshelly1 on 28 October 2013 - 03:21 PM in Dissertation and Paper Writing

When you go to endnote in word > search references > the external endnote program opens > do you see a drop down menu with formatting styles? I have version X5. You can also find this info in edit > output styles.

 

Good?




#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?




#162082 Suppression of a gene by Antisense strategy

Posted by jerryshelly1 on 04 November 2013 - 08:35 AM in Molecular Biology

Are you saying that you want to clone your gene of interest into a vector that will allow for suppression of your gene?

 

Edit - That is confusing. Are you saying that you want to clone your gene into a vector, in the REVERSE direction with the hopes that it will hybridize and suppress your mRNA?




#162112 Suppression of a gene by Antisense strategy

Posted by jerryshelly1 on 05 November 2013 - 08:16 AM in Molecular Biology

This isn't as simple as you would imagine. Researchers pay a decent amount of money for companies to manufacture antisense oligos for gene suppression. Usually when you request antisense oligo's, the manufacturer will send you multiple different plasmids containing different oligo sequences that could potentially disrupt gene expression. As you would expect, it is difficult to determine the correct anti-sense sequence that will disrupt gene function and multiple need to be used to determine which sequence can convey the greatest degree of supression.

 

If you are serious about doing this yourself, you should scan all the relevant literature and try to find a group who has successfully "knocked down" your gene of interest and request the oligo sequence that accomplished this. You may even be able to request a small sample of their plasmid if the lab is feeling generous. I honestly wouldn't attempt this myself, unless I had a significant amount of free time and even then I do not think it is worth the time to develop these myself.




#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




#160720 Storage of Milli Q Water

Posted by jerryshelly1 on 27 September 2013 - 07:03 AM in General Lab Techniques

Just out of curiosity, why would you avoid the autoclave?




#160706 Storage of Milli Q Water

Posted by jerryshelly1 on 26 September 2013 - 06:19 PM in General Lab Techniques

Make sure the pyrex is clean, add MilliQ water and then autoclave it.  As long as you maintain a seal on the bottle, you could potentially store it indefinitely. Even when I am using my Autoclaved MilliQ water as a stock, I like to frequently rinse, add new MilliQ water and autoclave (once every two or so weeks). A clean Pyrex bottle should not leach.




#164510 storage of laboratory human blood samples(whole anticoagulated blood-serum-plasm

Posted by jerryshelly1 on 25 January 2014 - 10:41 AM in General Biology Discussion

Depends what type of assay you want to do with them. We store whole blood at 4C for about 3-5 days if we want to extract DNA. Any longer than that and the blood is moved to the -80C for long term storage. The storage conditions will vary depending on what you need from the blood.





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