1. Will be compatible with Agarose TAE gels, when loaded directly?
2. Will it be compatible in Phusion PCR reactions, further as template?
Note: The manufacturer recommends 6m GuHCL for elution of Biotin tagged dsDNA from these beads (Roche).
If the DNA is double-stranded, you should be able to elute out the single-stranded DNA without using GuHCl.
DNA bound to the beads can be used directly in a PCR reaction - but may require modification of the procedure.
I would contact Roche and ask them specifically.
They may have some other suggestions rather than GuHCl.
Hello James,
Thanks for your suggestion. I will contact Roche regarding this matter. But I would also like to provide some more details about this experiment so that you may get a better idea and also give me your opinion.
This is control experiment for a bigger experiment.
I use Biotin-labeled primers for a PCR reaction (only one primer has biotin). Now, I have too check if i can pull them down with Streptavidin beads. I have checked both steps binding and elution. For elution, I used water at 70 deg, 6M GuHCL ( in case of GuHCL, i purify the samples to elute them in water)
For checking if at all the DNA was bound, I added small volume of millipore water to the DNA bound beads and then heated it to 95 deg for 5 minutes, then immediately separated the solution using a magnetic rack. I loaded the samples onto a Formamide denaturing gel, where I was able to see bands. That means the DNA was bound to the beads and the elution was inefficient.
I hope you understood.
Regards,
Sudip
Sudip,
You "loaded the samples" - what exactly are the samples? Beads with DNA, or the eluted DNA? Or both?
One thing, post-PCR do you do anything to remove any excess biotin-labelled primers remaining in the reaction. If this isn't done, then any of the remaining primers - or free floating biotin - may outcompete the PCR product for binding to the streptavidin.
If you want to check that any of the product is bound, then do a PCR on the product bound directly to the beads. This would be a very sensitive test to determine whether or not you actually bound any of the expected PCR product.
James
The sample means the eluted products and the supernatents after the bead separation on the magnet. I did not load any beads on to the gel.
And yes, I do purify using a kit after PCR to avoid any free biotin or remaining biotin-labeled primers. And then, I go for the pull down.
Hello Sudip,
what do you mean "with in case of GuHCL, i purify the samples to elute them in water".
If you are not going to reuse the beads you can elute your DNA with free-Biotin in mM range concentration. Depending from what is your contaminant you can also use desalting columns, Ethanol Precipitation and Ethyl Acetate Extraction to change the buffer after the elution step.
I'd like to ask more detail about your experiment with heating at 95 and then the denaturing gel: have you done this after the elution step? If yes, was the concentration compatible with a PCR reaction? What about your eluted fraction on the gel?
Best,
Francesco
Hello Francesco,
I am working with NGS sequencing and we have a customized protocol serving our purpose. The last step in the protocol is a Nested PCR. The PCR-1 is uses Biotin labeled primers, which can be exploited to enrich only specific products from a mixture. The Streptavidin pull down is used to enrich these specific amplicons, which will serve as template for the PCR-2.
Before doing this, I wanted make sure my Streptavidin pull down works. For this purpose, I used Biotin-labeled primers for a known sequence and amplified with PCR.
Now, I am supposed to have all amplicons labeled with Biotin. This would help me in quantifying the percentage recovery. Since, I used Streptavidin Magnetic Particles from Roche, I followed their recommendation of eluting the bound DNA with 6m Guanidine HCL.
I thought may be the GuHCL is hindering the proper running of Agarose gel so, I purified the sample using a PCR purification kit and eluted in water ( Ans to your Ques: with in case of GuHCL, i purify the samples to elute them in water".) And, then use this sample to visualize on Agarose gel.
But doing so also did not yield any recovery, which prompted me to subject the beads from the experiment to 95 degrees in water and then separate the beads on a magnet. I used the supernatent from this step to load it onto a Formaldehyde gel with MOPS buffer, usually used for ssRNA anaylsis. The motive behind using this being the fact that the 95 degree step will release ssDNA which could be immediately visualized on this type of gel. To my surprise, I found a good specific band on this gel conveying that there was a lot of DNA still bound to the beads and the elution with 6M GuHCL was not efficient.
I really liked your idea of using free Biotin, could you suggest me a protocol?
Regards,
Sudip
Hello Sudip,
I see that the Roche Streptavidin beads have a capacity of 1800 pmol/mg for free-biotin. I suggest to load three times, or more, this amount (it depends how much beads you used) then you can take the supernatant. I don't think the free biotin can inhibit PCR, but you can also remove it.
Alternatively, you can try the invitrogen protocols for the Elution.
Best,
Francesco
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