Comments for MO-BIO: The Culture Dish http://www.mobio.com/blog Thu, 16 May 2013 10:57:37 +0000 http://wordpress.org/?v=2.9.1 hourly 1 Comment on A Quick Guide for Troubleshooting Problems with PCR Amplification by Elena http://www.mobio.com/blog/2011/03/01/a-quick-guide-for-troubleshooting-problems-with-pcr-amplification/comment-page-1/#comment-14542 Elena Thu, 16 May 2013 10:57:37 +0000 http://www.mobio.com/blog/?p=1761#comment-14542 Dear Suzanne, thank you for your useful post. I am working on difficult samples (biopsies from which I have to amplify bacterial DNA, which I know it's going to be at low concentration). I'm having problems in PCR, probably due to inhibitors (mucus polysaccharides or something like this... I have verified this by adding a single strain genomic DNA that I knew can amplify to the bioptic DNA, and PCR does not work well on it). I tried to change some parameters in my extraction protocols but the problems stays. Can you tell me who should I contact to see if it is possible to try the MoBio PowerClean Kit to post-purify a couple of samples and see if the problems disappeared? I would not like to purchase the whole kit without knowing if it can be of any help on my samples... do you think it will be possible to receive a sample to test the efficacy on my samples? thank you in advance for your help. Elena Dear Suzanne, thank you for your useful post. I am working on difficult samples (biopsies from which I have to amplify bacterial DNA, which I know it’s going to be at low concentration). I’m having problems in PCR, probably due to inhibitors (mucus polysaccharides or something like this… I have verified this by adding a single strain genomic DNA that I knew can amplify to the bioptic DNA, and PCR does not work well on it). I tried to change some parameters in my extraction protocols but the problems stays. Can you tell me who should I contact to see if it is possible to try the MoBio PowerClean Kit to post-purify a couple of samples and see if the problems disappeared? I would not like to purchase the whole kit without knowing if it can be of any help on my samples… do you think it will be possible to receive a sample to test the efficacy on my samples? thank you in advance for your help.
Elena

]]> Comment on Sterivex Water Filter DNA Extraction? No Problem! by Richa http://www.mobio.com/blog/2011/06/03/sterivex-water-filter-dna-extraction-no-problem/comment-page-1/#comment-14540 Richa Thu, 16 May 2013 08:30:09 +0000 http://www.mobio.com/blog/?p=1899#comment-14540 Hi Suzanne, Thanks for your suggestion. I am using sterivex filters [Millipore, SVGV 010RS]. I have been using parafilm to prevent leakage. But can parafilm withstand 56 degrees overnight as I have to incubate sterivex at this temperature overnight ? looking forward for your suggestion. Richa Hi Suzanne,

Thanks for your suggestion.
I am using sterivex filters [Millipore, SVGV 010RS].

I have been using parafilm to prevent leakage. But can parafilm withstand 56 degrees overnight as I have to incubate sterivex at this temperature overnight ?

looking forward for your suggestion.

Richa

]]> Comment on Sterivex Water Filter DNA Extraction? No Problem! by Richa http://www.mobio.com/blog/2011/06/03/sterivex-water-filter-dna-extraction-no-problem/comment-page-1/#comment-14532 Richa Wed, 15 May 2013 13:47:12 +0000 http://www.mobio.com/blog/?p=1899#comment-14532 Hi, Could you Please suggest any cap for sterivex in order to to pack it from both sides ? Richa Hi,

Could you Please suggest any cap for sterivex in order to to pack it from both sides ?

Richa

]]> Comment on Homogenization Tips: Choosing a Bead Tube by Nanda http://www.mobio.com/blog/2010/08/13/homogenization-tips-choosing-a-bead-tube/comment-page-1/#comment-14507 Nanda Fri, 10 May 2013 21:19:47 +0000 http://www.mobio.com/blog/?p=1347#comment-14507 Hello, I want to use the 0.7 mm garnet to isolate RNA from larvae. How long should I sit the tube on the vortex at what setting? Also do the tubes go into microcentrifuge? Thanks! Hello,

I want to use the 0.7 mm garnet to isolate RNA from larvae. How long should I sit the tube on the vortex at what setting? Also do the tubes go into microcentrifuge?

Thanks!

]]> Comment on Homogenization Tips: Choosing a Bead Tube by Cherry http://www.mobio.com/blog/2010/08/13/homogenization-tips-choosing-a-bead-tube/comment-page-1/#comment-14500 Cherry Fri, 10 May 2013 08:39:01 +0000 http://www.mobio.com/blog/?p=1347#comment-14500 Dear Suzanne, I have a Zymo Tissue and insect RNA mini prep with me. It has bashing bead lysis tubes. My one problem is that i don't have the beadbeater or gene disruptor. Will it be okay if i were to use a vortex for homogenization and then go for high speed centrifugation? Or is there any other way i can proceed with since i need to carry out RNA isolation from a single mosquito in a few days. .Would appreciate your comments on this. Thank you Dear Suzanne, I have a Zymo Tissue and insect RNA mini prep with me. It has bashing bead lysis tubes. My one problem is that i don’t have the beadbeater or gene disruptor. Will it be okay if i were to use a vortex for homogenization and then go for high speed centrifugation? Or is there any other way i can proceed with since i need to carry out RNA isolation from a single mosquito in a few days. .Would appreciate your comments on this. Thank you

]]> Comment on A Quick Guide for Troubleshooting Problems with PCR Amplification by Suzanne Kennedy http://www.mobio.com/blog/2011/03/01/a-quick-guide-for-troubleshooting-problems-with-pcr-amplification/comment-page-1/#comment-14492 Suzanne Kennedy Wed, 08 May 2013 19:42:41 +0000 http://www.mobio.com/blog/?p=1761#comment-14492 Hi Rimi, Many thanks for reading out blog and for your question. If you don't want to dilute the DNA, you can try adding BSA. Some people have had success with this. Adding 0.5 ug/ul of BSA can help overcome inhibition. We do have a PowerClean Kit which can remove environmental inhibitors very fast and easy. I can send samples to you if you want to try it. I can also include samples of our PowerLyzer PowerSoil Kit to use for the next time to prepare genomic DNA from the sediments. This should do the trick. Let me know if you want samples and send an address for shipping and I can organize that for you. Best, Suzanne Hi Rimi,
Many thanks for reading out blog and for your question.
If you don’t want to dilute the DNA, you can try adding BSA. Some people have had success with this.
Adding 0.5 ug/ul of BSA can help overcome inhibition.
We do have a PowerClean Kit which can remove environmental inhibitors very fast and easy. I can send samples to you if you want to try it.
I can also include samples of our PowerLyzer PowerSoil Kit to use for the next time to prepare genomic DNA from the sediments. This should do the trick.

Let me know if you want samples and send an address for shipping and I can organize that for you.
Best,
Suzanne

]]> Comment on A Quick Guide for Troubleshooting Problems with PCR Amplification by Suzanne Kennedy http://www.mobio.com/blog/2011/03/01/a-quick-guide-for-troubleshooting-problems-with-pcr-amplification/comment-page-1/#comment-14491 Suzanne Kennedy Wed, 08 May 2013 19:38:42 +0000 http://www.mobio.com/blog/?p=1761#comment-14491 Hi Britt, Thanks for your email and I'm glad you found the blog article. OK, I want to be sure I understand. You've done end-point PCR before but not qPCR (qPCR = quantitative PCR and qRT-PCR = quantitative reverse transcription PCR). SYBR Green is a dye and Taqman is a probe- you used a SYBR Green Kit? Whose kit are you using? OK- a couple things. First, make sure the amplification product is small- below 250 bp. Ideally the smaller the better. The qPCR chemistry is designed for fast cycling and the PCR product needs to be small. Did you use the cycling conditions recommended by the kit supplier? The cycling for end-point PCR and SYBR Green PCR is different. Next, on the machine, you have to set it and tell it when to collect data. For SYBR Green, this is typically during the extension phase if you are doing three step cycling (95-60-72C). You may need to turn on or activate the sensors to collect data. Whose qPCR machine do you have? Let's start here and then I think I can help you get this right. Best, Suzanne Hi Britt,
Thanks for your email and I’m glad you found the blog article.
OK, I want to be sure I understand. You’ve done end-point PCR before but not qPCR (qPCR = quantitative PCR and qRT-PCR = quantitative reverse transcription PCR). SYBR Green is a dye and Taqman is a probe- you used a SYBR Green Kit? Whose kit are you using?

OK- a couple things. First, make sure the amplification product is small- below 250 bp. Ideally the smaller the better. The qPCR chemistry is designed for fast cycling and the PCR product needs to be small.
Did you use the cycling conditions recommended by the kit supplier? The cycling for end-point PCR and SYBR Green PCR is different.
Next, on the machine, you have to set it and tell it when to collect data. For SYBR Green, this is typically during the extension phase if you are doing three step cycling (95-60-72C). You may need to turn on or activate the sensors to collect data.
Whose qPCR machine do you have?

Let’s start here and then I think I can help you get this right.
Best,
Suzanne

]]> Comment on Molecular Biology of Soil: DNA Isolation Part I by Larai http://www.mobio.com/blog/2010/01/17/molecular-biology-of-soil-dna-isolation-part-i/comment-page-1/#comment-14477 Larai Tue, 07 May 2013 12:08:23 +0000 http://www.mobio.com/blog/?p=596#comment-14477 I have used the power soil DNA extraction kit on a number for soil (rhizosphere) samples (a bit moist) and checked the yields on Nanodrop. I have a yeild ranging from 1.7 to 5.1ng/ul. The 260/280 ratio for the 1.7ng/ul is 2.36 while the 260/230 ratio is 10.48. The one with the 5.2ng/ul has 260/280=1.40 and 260/230=0.85. I was actually expecting better yields but I am worried about the down stream application (PCR and fragment analysis). Are these yeilds sufficient? if not how can I get better yields? Please I would appreciate some technical advice. I have used the power soil DNA extraction kit on a number for soil (rhizosphere) samples (a bit moist) and checked the yields on Nanodrop. I have a yeild ranging from 1.7 to 5.1ng/ul. The 260/280 ratio for the 1.7ng/ul is 2.36 while the 260/230 ratio is 10.48. The one with the 5.2ng/ul has 260/280=1.40 and 260/230=0.85.

I was actually expecting better yields but I am worried about the down stream application (PCR and fragment analysis). Are these yeilds sufficient? if not how can I get better yields?

Please I would appreciate some technical advice.

]]> Comment on A Quick Guide for Troubleshooting Problems with PCR Amplification by rini http://www.mobio.com/blog/2011/03/01/a-quick-guide-for-troubleshooting-problems-with-pcr-amplification/comment-page-1/#comment-14475 rini Tue, 07 May 2013 07:49:10 +0000 http://www.mobio.com/blog/?p=1761#comment-14475 hai suzanne I have checked the purity and concentration of my RNA of a plant sample. I have a very high concentrated RNA but the purity is somewhere around 1.3 to 1.4...so is there anything i can do to increase the purity of my RNA sample? I think we can do proteinase-K treatment but how exactly I doesnty know.can you give me some protocol links for that...is it ok if we do the proteinase treatment after extraction of RNA?are there chances that my RNA may get degraded during the process?please tell me as early as possible..thank you hai suzanne
I have checked the purity and concentration of my RNA of a plant sample. I have a very high concentrated RNA but the purity is somewhere around 1.3 to 1.4…so is there anything i can do to increase the purity of my RNA sample? I think we can do proteinase-K treatment but how exactly I doesnty know.can you give me some protocol links for that…is it ok if we do the proteinase treatment after extraction of RNA?are there chances that my RNA may get degraded during the process?please tell me as early as possible..thank you

]]> Comment on A Quick Guide for Troubleshooting Problems with PCR Amplification by Rimi http://www.mobio.com/blog/2011/03/01/a-quick-guide-for-troubleshooting-problems-with-pcr-amplification/comment-page-1/#comment-14470 Rimi Mon, 06 May 2013 03:28:58 +0000 http://www.mobio.com/blog/?p=1761#comment-14470 Hi Suzanne, I am working on metagenomics of sediment samples. Few sediment DNA samples have failed to amplify by using 16S primers. Probably they contain enzyme inhibitors. Instead of diluting the DNA, do we have any other alternatives? Please reply soon. Regards, Rimi Hi Suzanne,

I am working on metagenomics of sediment samples. Few sediment DNA samples have failed to amplify by using 16S primers. Probably they contain enzyme inhibitors. Instead of diluting the DNA, do we have any other alternatives? Please reply soon.

Regards,
Rimi

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