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DNA and General PCR Methods :: Macrogen - server problems?

Author: smoo
Subject: Macrogen - server problems?
Posted: Jul 03 2008 4:38 pm (GMT -4)
Topic Replies: 0

Just wondering if anyone else uses the Macrogen sequencing service in Korea and is having problems logging in at the moment. Earlier today the server seemed to be down and now my login ID is no longer recognized. I have some results to collect. I've emailed them, but if this is a universal problem I guess they're going to be very busy and unlikely to respond quickly.

Real-Time qPCR/qRT-PCR Methods :: RE: cDNA dilution but no change in Ct values

Author: mchlbrmn
Posted: Jul 03 2008 1:34 pm (GMT -4)
Topic Replies: 3

Ct means "crossing threshold" (I think). This means the cycle number it takes to product enough fluorescence to reach an arbitrary threshold (may be determined in different ways). That should explain why higher Ct is less starting template. (You get fractions of a cycle because the computer is smart and does extr0polations (sp?) for you).
Ct can be artificially high if you use SYBR green, which "sees" everything, and have non-specific PCR products showing up. This may be more likely to happen in later cycles. The melting peak analysis after the cycling can tell you (sometimes) if undesired products are being generated. So, if you have no non-specific stuff until after, say, 32 cycles, and lots after, that would make less diffence in cycles than predicted by the dilutions (and make the PCR efficiency appear too high).

Humor in Science :: Amateur Transplants

Author: Katzenjammer
Subject: Amateur Transplants
Posted: Jul 03 2008 1:30 pm (GMT -4)
Topic Replies: 0

This isn�t really about science, but in my opinion it�s very good song

http://www.youtube.com/watch?v=xuZl9tRqjoQ&feature=related

and something about drugs, try remember all words:

http://www.youtube.com/watch?v=69dGvr-asP4&feature=related

I'm new here by the way.

Cell Culture :: PBS - where are my cells?

Author: Lis
Subject: PBS - where are my cells?
Posted: Jul 03 2008 1:14 pm (GMT -4)
Topic Replies: 0

Hi everyone,

I have a problem! I've been testing pre-treatment of collagen substrates related to cell attachment and proliferation.

Basically, I soaked overnight 3 different collagen substrates in PBS and fibroblast medium (separately) and used non-soaked substrates as controls.
Fibroblasts were seeded on the surface of these substrates (5x10^5 cells/cm2), and incubated with fibroblast medium for 28 days.
At each medium change i checked the old medium for dead and viable cells (trypan blue) and performed MTT test.

At the end of the study i checked the plastic surface for fibroblast attachment and performed histology in the collagen substrates to look for cell attachment and infiltration.

2 of the substrates soaked in PBS showed no fibroblasts, not even in the plastic surface of the wells. I did not found more dead/viable cells on the "old medium" throughout the experiment (compared to the values i obtained from the ones soaked in fibroblast medium or the controls).

Have anyone had problems with PBS before?
Will it make my substrate unfriendly to the medium proteins and therefore, not allow cell attachment?
Does PBS cause cell death?

Any references in this subject?

Many thanks!!
_________________
LCB

Real-Time qPCR/qRT-PCR Methods :: RE: cDNA dilution but no change in Ct values

Author: aliblain
Posted: Jul 03 2008 11:56 am (GMT -4)
Topic Replies: 3

You should expect a change of 1 Ct for every doubling of template- so there is a big difference between 30 and 36 cycles.

Real-Time qPCR/qRT-PCR Methods :: RE: Triplicates- to remove outliers or not...

Author: aliblain
Posted: Jul 03 2008 11:51 am (GMT -4)
Topic Replies: 8

I would agree that triplicates are useful in reducing the effect of pipetting error. Sudders- I assessed pipetting error during optimisation of my experiment using a plate of identical samples with cDNA added separately to each well.

DNA and General PCR Methods :: RE: inserting 120bp DNA

Author: mchlbrmn
Posted: Jul 03 2008 11:09 am (GMT -4)
Topic Replies: 2

What were you thinking of: making a PCR product with the ends homologous to the vector (homologous Tm of each end >65), purifying, and using the PCR product as "primers" for the "quickchange" reaction? That sounds good to me as long as there's not enough homology so the huge "primers" (PCR product strands) land elsewhere in the vector or have too much secondary structure within the "primers". I don't know which Quickchange is best, but I guess any kit with a polymerase that can handle a final vector of size you have (or any proofreading polymerase without the kit, using the same protocol). That's probably quickest, but there's a chance it won't work, so restriction enz. cloning/ligation is more certain to work first time, I guess.

DNA and General PCR Methods :: RE: Specificity of a Northern hybrization?

Author: mchlbrmn
Posted: Jul 03 2008 10:55 am (GMT -4)
Topic Replies: 2

relaxin wrote:

If the transcripts of the two genes are of different sizes, then the full-length probe can be used without problem. If they are the same size, then more specific probe and high stringency washing will be needed.
70% sequence homology is very significant, especially the homology is concentrated in one location.

I don't know the sizes, there's no Northern published, and only one paper on these genes. The CDS are about the same, but the full Genbank reference sequences have different sizes, however. I read in a Northern protocol that indicates it's not recommended to have > 1kb probe as that increases the size of non-specific hybridization. All the more reason to avoid the homologous part of the CDS.
I ordered an primer cutting off the homologous part of the gene. I can make a smaller hot probe with this primer useing my existing 1.7 kb fragment as template with either klenow or maybe assymetric PCR (I suppose that's a bit nicer, but I should check that the PCR works well first without hot label and run on a gel).
Thanks for the response Relaxin.

Real-Time qPCR/qRT-PCR Methods :: EMA treatment

Author: kralikp
Subject: EMA treatment
Posted: Jul 03 2008 8:14 am (GMT -4)
Topic Replies: 0

Hello all,

we are trying to solve quite tricky problem. We try to introduce EMA or PMA-PCR for the determination of viable and dead cells. We started to optimize the treatments with different concentrationa s and exposure times on the isolated DNA, but there is still a positive signal in the qPCR althought all naked DNA should be inactivated for the PCR by intercalation of the dye to DNA. We use 50 ng of DNA in the total volume of 50 ul. Different concentrations of EMA and PMA are then added and subsequently after the treatment DNA is 10-fold diluted and 5 ul is added to the 20 ul of the qPCR reaction.

Does anybody of you have some experince with these chemicals when they are used for the live/dead cell determination?

Thank you in advance for suggestions.

Petr

DNA and General PCR Methods :: RE: Anybody has the following primer sequences?

Author: coddlecodd
Posted: Jul 03 2008 5:41 am (GMT -4)
Topic Replies: 3

Big Ploblem: pseudogenes.

Sorry my bad,
however if you run a test of genomic DNA contamination with your qPCRs (just any primers that amplify gDNA) at what CT values do you get a product? Using the on-filter DNase treatment I've never seen genomic contamination giving rise to high enough CTs to interfere with my actual genes. Are the genes poorly expressed?

/Joakim

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Author: xmojj
Subject: hi,everybody
Posted: Jul 03 2008 12:01 am (GMT -4)
Topic Replies: 0

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DNA and General PCR Methods :: RE: inserting 120bp DNA

Author: relaxin
Posted: Jul 02 2008 8:56 pm (GMT -4)
Topic Replies: 2

If you can copy the 120 bp by PCR using primers with built-in restriction sites, then it will be easy to subclone into a number of different vectors. If the restriction sites are different for different vector, just make new primers.
_________________
Not affiliated with any company. Mentioning of a specifc product does not imply my endorsement of the product to the exclusion of other equally good ones.

DNA and General PCR Methods :: inserting 120bp DNA

Author: bio22
Subject: inserting 120bp DNA
Posted: Jul 02 2008 7:23 pm (GMT -4)
Topic Replies: 2

What would be the easiest way to insert 120bp into a vector? Would the Quickchange XL kit from Stratagene be able to handle that size of insert?

Also, I may have to make a few new vectors with this 120bp tail, what would be the easiest way to get it into my PCR product?

RNA Methods :: RE: 30% glycerol stock preparation

Author: relaxin
Posted: Jul 02 2008 5:47 pm (GMT -4)
Topic Replies: 1

Just add 30 ml of glycerol in a graduated cylinder, fill up to 100 ml with deionized water. Seal the cylinder with parafilm, and mix by inversion. Transfer to a bottle and autoclave for 20 min in liquid cycle.
_________________
Not affiliated with any company. Mentioning of a specifc product does not imply my endorsement of the product to the exclusion of other equally good ones.

DNA and General PCR Methods :: RE: Specificity of a Northern hybrization?

Author: relaxin
Posted: Jul 02 2008 5:41 pm (GMT -4)
Topic Replies: 2

If the transcripts of the two genes are of different sizes, then the full-length probe can be used without problem. If they are the same size, then more specific probe and high stringency washing will be needed.
70% sequence homology is very significant, especially the homology is concentrated in one location.
_________________
Not affiliated with any company. Mentioning of a specifc product does not imply my endorsement of the product to the exclusion of other equally good ones.