General Lab Techniques - BioWiki Knowledge Base http://www.protocol-online.org/forums/page/index.html/_/generallabtechniques/ Fri, 10 Feb 2012 17:35:32 +0000 43200 DNA migrate differently in agarose vs PAGE gel http://www.protocol-online.org/forums/page/index.html/_/generallabtechniques/electrophoresis/dna-migrate-differently-in-agarose-vs-page-gel-r20 Quote

i am currently working a gene in leukemia. The mutant allele has been sequenced to contain 527 base pair. when i run an agarose gel to confirm, it did appear in the position approximately equal to 527 base pair according to the DNA ladder. HOWEVER, when i run a PAGE, it shows up at a different poistion!!(it is at about 600 base pair in PAGE!!!!). Yet the conditions were the same when i run the PAGE and agarose gel (150v, 30 mins)


Many things can affect DNA migration in gel. Check this page "Top 10 Fun Facts for DNA Electrophoresis" to see if there is anything that might have caused the abnormal migration. For example "On a polyacrylamide gel, DNA fragments having AT-rich regions migrate slower than other DNA fragments of the same size."

According to Fermentas troubleshooting, here is a list of things that may affect migration of DNA in gel:

Atypical migration due to different DNA sequence or structure. During high resolution electrophoresis DNA fragments of equal size can migrate differently due to differences
in DNA sequences. AT rich DNA may migrate slower than an equivalent size GC rich DNA fragment. DNA structures such as nicked, supercoiled or dimeric molecules will always show different mobility on gels compared to an equivalent DNA size standard.

Gel shift effect. The presence of DNA binding proteins in the sample, such as ligases, phosphatases or restriction enzymes may alter DNA migration in the gel or cause the DNA to remain in the gel wells. High salt concentration in the sample may also cause gel shift effects.]]> Tue, 16 Aug 2011 07:28:19 +0000 da4fb5c6e93e74d3df8527599fa62642 Normalization for RNA or cDNA during two step R... http://www.protocol-online.org/forums/page/index.html/_/generallabtechniques/normalization-for-rna-or-cdna-during-two-step-r-r14 Curtis 20 April 2009 - 03:13 AM
Hello all,

I have a two-step SYBR Green RT-PCR kit for RotorGene. After I extract RNA from my samples, I proceed with cDNA synthesis right away without measuring the RNA concentration or without normalizing equal amount of RNA for all my samples. It is only after making cDNA that I measure the concentration and add around 100 ng in each PCR tube.

my results are a bit funny now and I get my first signal after 35 cycles and surprisingly the samples that should give signal sooner are always the last ones. my friends are suggesting to measure my RNA before proceeding with cDNA synthesis. They say maybe too much RNA in the sample does not let cDNA to be properly synthesized !?

is this comment true? Reply Edit
public/style_images/mobile/profile/default_thumb.png littleaxt 20 April 2009 - 05:42 AM
Hi

How do you measure cDNA concentration? Do you purify it before measureing it? Because I was told that if I measure with the Nanodrop I would also measure the unused dNTPs and therefore I could also start making it up :-)...


public/style_images/mobile/snapback.pngCurtis, on Apr 20 2009, 12:13 PM, said:

Hello all,

I have a two-step SYBR Green RT-PCR kit for RotorGene. After I extract RNA from my samples, I proceed with cDNA synthesis right away without measuring the RNA concentration or without normalizing equal amount of RNA for all my samples. It is only after making cDNA that I measure the concentration and add around 100 ng in each PCR tube.

my results are a bit funny now and I get my first signal after 35 cycles and surprisingly the samples that should give signal sooner are always the last ones. my friends are suggesting to measure my RNA before proceeding with cDNA synthesis. They say maybe too much RNA in the sample does not let cDNA to be properly synthesized !?

is this comment true? Reply Edit Delete
uploads/profile/photo-thumb-6817.jpg Curtis 20 April 2009 - 06:45 AM
good point, but if I purify the cDNa I'm gonna lose a lot of it during the purification steps. after all we are not talking about a PCR product that has been amplified. it's just cDNA from our RNA which is also not a lot. Reply Edit Delete
public/style_images/mobile/profile/default_thumb.png gfischer 20 April 2009 - 07:45 AM
I always normalize the amount of RNA I use in my RT reaction. This way, you know that the only source of variation in the amount of signal in the quantitative step is from a higher level of relative expression of target RNA. Reply Edit Delete
public/style_images/mobile/profile/default_thumb.png littleaxt 20 April 2009 - 08:48 AM
Oh, it seems possible, see http://www.biomedcen.../1471-2199/9/18 they also did it. Seems like cDNA clean-up also enhances the PCR.

But I never tried it myself, I normalize against total RNA prior reverse transcription and use relative quantification.

All the best

Jan

public/style_images/mobile/snapback.pngCurtis, on Apr 20 2009, 03:45 PM, said:

good point, but if I purify the cDNa I'm gonna lose a lot of it during the purification steps. after all we are not talking about a PCR product that has been amplified. it's just cDNA from our RNA which is also not a lot. Reply Edit Delete
uploads/profile/photo-thumb-6817.jpg Curtis 20 April 2009 - 09:07 AM
littleaxt: yeah, relative quantification. that's what I'm doing now. the link you gave doesn't open for me.

gfischer: that would be for one-step RT-PCR? or applies to two-step as well? how do you make sure you won't have more than 100 ng of cDNA in your sample? because if you have more than 100 ng the amplification might not work properly. Reply Edit Delete
public/style_images/mobile/profile/default_thumb.png littleaxt 21 April 2009 - 12:04 AM
ok, this might help:

Libus & Štorchová 2006 “Quantification of cDNA generated by reverse transcription of total RNA provides a simple alternative tool for quantitative RT-PCR normalization” BioTechniques 41, 156-164

Liss 2002 “Improved quantitative real-time RT-PCR for expression profiling of individual cells” Nucleic Acids Research 30 (17): e89

Hibbeler et al. 2008 “Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus“ BMC Molecular Biology 8:18

First is another idea you could try, second is a precipitation protocol for cDNA (never tried it myself) third is another paper where they made a cDNA clean-up prior PCR. Hope it helps, good luck!


public/style_images/mobile/snapback.pngCurtis, on Apr 20 2009, 06:07 PM, said:

littleaxt: yeah, relative quantification. that's what I'm doing now. the link you gave doesn't open for me.

gfischer: that would be for one-step RT-PCR? or applies to two-step as well? how do you make sure you won't have more than 100 ng of cDNA in your sample? because if you have more than 100 ng the amplification might not work properly. Reply Edit Delete
uploads/profile/photo-thumb-6817.jpg Curtis 21 April 2009 - 07:08 AM
ok thank you, I'll have a look now Reply Edit Delete
public/style_images/mobile/profile/default_thumb.png Trof 22 April 2009 - 04:33 AM
We do two-step RT real-time PCR and always measure the amount of RNA only. Usually we put 1-3 ug of RNA to the RT reaction, depending how much cDNA we need. We don't measure nor clean-up the cDNA, use it directly to the real-time PCR.
We can dilute the cDNA if we need more volume for more samples (like 2 times or 3 times), but usually only when using more than 1 ug. Never exceed the 10 % of cDNA in a reaction mix. That makes from 1 ug in 20ul RT mix, when using maximum 2ul to a 20ul PCR reaction just about 100 ng.

I think we would see it on the aplification curve if there is any problem with high amount of cDNA, we never encountered such thing. Reply Edit Delete
uploads/profile/photo-thumb-6817.jpg Curtis 23 April 2009 - 10:18 PM
public/style_images/mobile/snapback.pngTrof, on Apr 22 2009, 03:33 AM, said:

We do two-step RT real-time PCR and always measure the amount of RNA only. Usually we put 1-3 ug of RNA to the RT reaction, depending how much cDNA we need. We don't measure nor clean-up the cDNA, use it directly to the real-time PCR.
We can dilute the cDNA if we need more volume for more samples (like 2 times or 3 times), but usually only when using more than 1 ug. Never exceed the 10 % of cDNA in a reaction mix. That makes from 1 ug in 20ul RT mix, when using maximum 2ul to a 20ul PCR reaction just about 100 ng.

I think we would see it on the aplification curve if there is any problem with high amount of cDNA, we never encountered such thing.

Thank you Trof,
your explanation was very helpful. Reply Edit Delete
uploads/profile/photo-thumb-10294.jpg Chimp 16 June 2009 - 12:54 AM
Checking [RNA] is an important step. You could also run a denaturing RNA gel to check RNA integrity, you should get clear rRNA bands Reply Edit Delete
public/style_images/mobile/profile/default_thumb.png Freddynb 02 September 2009 - 05:43 AM
I have developed a method to amplify multiple random genes in a single real-time RT-PCR reaction to assess quality of cDNA between samples. It can be used to normalize gene expression between samples. Please see
http://www.springerl...28327382188710/ Reply Edit Delete
public/style_images/mobile/profile/default_thumb.png sssbio 29 May 2010 - 03:32 PM
public/style_images/mobile/snapback.pngFreddynb, on Sep 2 2009, 01:43 PM, said:

I have developed a method to amplify multiple random genes in a single real-time RT-PCR reaction to assess quality of cDNA between samples. It can be used to normalize gene expression between samples. Please see
http://www.springerl...28327382188710/

Hi,

I need your help regarding quantification of mRNA levels of trageted genes in control and treated samples. I have no choice of reference housekeeping genes to be used for relative quantification as these genes are showing significant down regulation in treated samples. So what are other methods that I can use to quantify mRNA levels?

I will be very happy if you guide me for this.

Thanks Reply Edit Delete
public/style_images/mobile/profile/default_thumb.png gradechips 02 June 2010 - 11:05 PM
Please help me, I have been doing RT-PCR for the past six months and i still have problems. During this period i was only able to get decent Cts for the house keeping gene (18) twice but all the other times the Cts for both GAPDH/beta actin have been really high 25 or inconsistent within triplicate samples (20, 24, 29). Obviously i cant use these data to analyze gene expression and am getting frustrated. After extracting RNA using Trizol i quantify it by ODing and i usually get ~2.500ng/ul and i use 2ug for the cDNA synthesis and i normally dilute the cDNA 1:50. My lab mate normally gets low and consistent Ct for the house keeping gene using the same reagents. I have been trouble shooting for the past 6 months with his help but am not getting it.
Is it because vortexing shear my samples? Please someone help me.
Thanks in advance Reply Edit Delete
public/style_images/mobile/profile/default_thumb.png Nataliya 24 June 2010 - 01:19 AM
public/style_images/mobile/snapback.pnggradechips, on Jun 2 2010, 11:05 PM, said:

Please help me, I have been doing RT-PCR for the past six months and i still have problems. During this period i was only able to get decent Cts for the house keeping gene (18) twice but all the other times the Cts for both GAPDH/beta actin have been really high 25 or inconsistent within triplicate samples (20, 24, 29). Obviously i cant use these data to analyze gene expression and am getting frustrated. After extracting RNA using Trizol i quantify it by ODing and i usually get ~2.500ng/ul and i use 2ug for the cDNA synthesis and i normally dilute the cDNA 1:50. My lab mate normally gets low and consistent Ct for the house keeping gene using the same reagents. I have been trouble shooting for the past 6 months with his help but am not getting it.
Is it because vortexing shear my samples? Please someone help me.
Thanks in advance


I usually use RNeasy Mini Kit (50) and RNase-Free DNase Set (50) from Qiagen. Once I have got RNA extracted I use it for cDNA synthesis or keep at -70 C. For cDNA synthesis I used SuperScriptTM III First-Strand Synthesis System for RT-PCR (Invitrogen, Cat. No: 18080-051). For RT-PCR I diluted cDNA 1:5. Sometimes I measured cDNA by nanodrop and the concentration of the diluted cDNA samples was around 200 ng/ul I have never faced the problem with low values for house-keeping genes. I normally get 13-14 Ct for GAPDH. Probably, you don't need to dilute samples up to 50 ul/ml.]]> Sat, 07 Aug 2010 20:44:51 +0000 5fd0b37cd7dbbb00f97ba6ce92bf5add <![CDATA[Useful Dilution Techniques & Calculations]]> http://www.protocol-online.org/forums/page/index.html/_/generallabtechniques/useful-dilution-techniques-calculations-r12 I noticed most of the students including myself had trouble with the dilution process. So I thought maybe I'd make a quick explanation for what is dilution and how it is done and all the ratios and other confusing stuff. I tried my best to simplify it for people who find trouble in doing their homeworks, labwork, or other calculation problems regarding dilution questions and solutions.

So let's start out with definitions:

Dilution: is the mixing of a small accurately measured sample with a large volume of sterile water or normal saline called (diluents or dilution blank)

Laws:

Dilution = V of Sample / Total V of (sample + diluent)

Dilution Factor = Total V of (sample + diluent) / V of sample
** or we can simply say the reciprocal of Dilution

Starting off with this simple example to understand how these laws are applied.


Posted Image


It has been known that if we use a larger volume we obtain a more accurate dilution.
So for better results, we use 1:1000 dilution. And that is by adding 1ml of sample to 999 ml of diluent. But practically we cannot use 999 ml of diluent. So we do what is called a serial dilution.

Serial Dilution: is a dilution made of a series of smaller dilution, and the total dilution is the product of each dilution in the series.

To understand this more, let's see this example.


Posted Image

Posted Image


Here's an example combining all of the above.


Posted Image


I hope this brief explanation proves helpful to you...let me know if i've made any mistakes... B)

-Property of Yulia-
thank u for your explanation..

what makes me always confused is the last volume used for inoculation (0.1ml in your example)..

-strawberry-

Yeah that's why i almost jumped with 'HAVE' there :blush: sorry about that
i hope my mini-tutorial and examples helped you through it strawberry...:)

-Property of Yulia-

I'm very thankful to you to provide the easier explaination...it's true that many undergrad students have problem in dilution..and the worst thing is if the lecturers/tutors assume that the students are familiar with the concept. it is true that they have learned it in high school, but most of the time they will forget..

Regarding the example 2 given, you dilute a sample (10 ml the final volume) 10x10x10=10 to the power of 3. and u inoculate 0.1 ml in your medium.so supposed the final volume of your medium is 1ml right?


-kent19-]]> Sat, 07 Aug 2010 05:55:42 +0000 7f6ffaa6bb0b408017b62254211691b5 How to convert g into rpm? http://www.protocol-online.org/forums/page/index.html/_/generallabtechniques/centrifugation/how-to-convert-g-into-rpm-r11 g = (1.118 × 10-5) R S2
where g is the relative centrifugal force, R is the radius of the rotor in centimeters, and S is the speed of the centrifuge in
revolutions per minute.

You can use this for any centrifuge, just measure the radius of the rotor from the center to outer edge.]]> Sat, 07 Aug 2010 05:54:42 +0000 698d51a19d8a121ce581499d7b701668