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



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#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.




#160879 I dissolved the DNA in 1XTAE instead of TE buffer, will it affect ligation?

Posted by jerryshelly1 on 03 October 2013 - 09:28 AM in General Lab Techniques

You should be fine.  I once used a sigma kit and they gave you an alternative ligation buffer if you suspended your DNA in TAE. When you are finished with your ligation, I would consider purifying your product somehow. TAE has been shown to significantly decrease the transformation efficiency of E. coli.

 

Google search pulled up the relevant kit with alternative buffer:

http://www.sigmaaldr...tmp/lig2bul.pdf




#160888 Ligation problem

Posted by jerryshelly1 on 03 October 2013 - 01:42 PM in Molecular Biology

What are your vector and insert sizes? Also, what are your incubation conditions? Did you use a control to verify your ligase is working?




#160892 Ligation problem

Posted by jerryshelly1 on 03 October 2013 - 02:04 PM in Molecular Biology

Sometimes if an restriction enzyme has been sitting unused for sometime, I will test each one by taking a known concentration of a plasmid and i will double check the efficiency of each enzyme (Add 5U of enzyme to whatever concentration of DNA and us HindIII ladder to see ng of linearized DNA). From here, digest my insert, CIP it and then ligate it for 16C for 2 hrs, 4C overnight, and 16C for 2 hrs. Depending on the quantity of my ligation mixture, I will either run it on a gel and gel extract my ligated product or just directly transfect it.

 

Good controls:

1) Plasmid without insert to verify activity of ligase.

2) Plasmid without insert and without ligase to check the activity of your CIP.

 

I usually use these controls to verify all of my enzymes are functional.

 

A good thing to check is your primer design.  Are you using an insert that was produced via PCR? Did you design the primers to produce an overhang that will provide maximum efficiency of your enzyme (reference - https://www.neb.com/...f-dna-fragments).

 

 

Does this help?




#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?




#160895 Ligation problem

Posted by jerryshelly1 on 03 October 2013 - 02:15 PM in Molecular Biology

You are correct. Everything sounds correct. Your PCR product was developed to have a sufficient overhang for digestion?

 

After you digested your plasmid, did you isolate your linearized product via gel purification?




#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?




#160919 Dimerization of PCR product

Posted by jerryshelly1 on 04 October 2013 - 09:04 AM in Molecular Cloning

Is this a transformation reaction from a previous ligation? Did you use two independent RE sites when you ligated your insert into your vector?

 

Colony PCR is notoriously bad because of its high generation of false positives.

 

Where are your primers annealing for your PCR reaction, i.e. both on plasmid, one plasmid + one insert? 




#161016 Repeated mutagensis primer in site-directed mutagenesis

Posted by jerryshelly1 on 08 October 2013 - 07:19 AM in PCR, RT-PCR and Real-Time PCR

Do you mean that you have two duplicates of your mutation in your gene? I am having trouble understanding what you mean.




#161035 Calcutation question

Posted by jerryshelly1 on 08 October 2013 - 12:27 PM in Tissue and Cell Culture

You just need to pick a total final volume and make all other calculations based on that. You want your total volume to be 1mL of media? Make all of your calculations based on 1mL of media and subtract the amount of stimulator A, B and C from your 1mL of media. 

 

Do you need to add your stimulators at different time points or are they all added at the exact same time.  If they are added at different time points, the 10.2uL difference will be negligible as the amount of stimulator A will have decreased due to your cells consuming it.

 

Does this make sense?




#161065 ABI 7300 qRT-PCR and ddCT protocol

Posted by jerryshelly1 on 09 October 2013 - 05:59 AM in PCR Reagents and Equipments

Academic institutions keeps receipts for ages.  If ABI is not willing to show you their original purchase receipt, find your institutions.  You can also usually get some leeway if you mention that you use there chemicals, plates, films for the machine and mention that many other companies make more suitable replacements. It has work for me with some things. I guess it just depends on the overall cost of the product.




#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...




#161098 Primer design

Posted by jerryshelly1 on 10 October 2013 - 07:23 AM in PCR, RT-PCR and Real-Time PCR

Are you using these primers to confirm the presence or absence of your coat protein? For your virus, does it matter if they primers can differentiate between PVY and PVA?




#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...




#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?




#161113 Primer design

Posted by jerryshelly1 on 10 October 2013 - 10:01 AM in PCR, RT-PCR and Real-Time PCR

it is possible, but are you looking at a collection of virus' or will your sample contain either PVY and PVA? If your sample contains only one of the virus', it will not be a problem.




#161138 Repeated mutagensis primer in site-directed mutagenesis

Posted by jerryshelly1 on 11 October 2013 - 07:16 AM in PCR, RT-PCR and Real-Time PCR

That is weird that you get perfect insertion of the primer multiple times.  One can see binding of the two primers when you run them through a dimer prediction program, but it doesn't match up with what you are seeing:

 

Thermo Scientific, Self-Dimers:

1 dimer for: primer
             5-aaacaaatttgttgattgggttctt->
              ||||||||||||
<-ttcttgggttagttgtttaaacaaa-5

1 dimer for: Primer
5-aagaacccaatcaacaaatttgttt->
              ||||||||||||
           <-tttgtttaaacaactaacccaagaa-5
 

 

Are the 5' and 3' regions of your insertion site similar that would allow multiple insertions?

 

Does the mutagenesis kit say that a 25bp primer is sufficient for a 2bp mutation? My protocols usually required >40bp for correct mutation.

 

Your results are weird to say the least.  Could you provide some sequence info on your 3' and 5' gene sequence?




#161139 Primer design

Posted by jerryshelly1 on 11 October 2013 - 07:19 AM in PCR, RT-PCR and Real-Time PCR

If you are studying the virus in a lab setting, chances are that only one variation of the virus (PVY or PVA) is present.  If only one is present, you only need one primer as any amplification you see would be from the coat protein of virus which is present.

 

Even so, are the PVY and PVA coat proteins 100% conserved?




#161143 Perculiar contamination of agarose gel combs?

Posted by jerryshelly1 on 11 October 2013 - 08:40 AM in General Lab Techniques

That is unusual.  Probably not from the combs (low retention).  Check your UV tray and make sure that an undergrad didn't crack the glass and leave EtBr residue within. Do you only see this pattern when a gel is present? It isn't in all your samples, so it probably isn't in your loading dye. It seems strange...




#161144 Control protein for eukaryotic expression

Posted by jerryshelly1 on 11 October 2013 - 08:43 AM in Protein Expression and Purification

HEK's are marvelous for expression studies. Whenever I have a problematic expression vector, I stick it into HEK's and see marvelous expression. HEK cells are a good choice.




#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. 




#161245 SNP discovery

Posted by jerryshelly1 on 14 October 2013 - 11:04 AM in Genetics and Genomics

All available human SNPs are on UCSC (hg19). If you have the DNA, you could sequence your genomic DNA.




#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.





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