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Old 08-15-2011, 11:57 PM   #1
g861@hotmail.com
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Default Same library to get different percentage of enriched beads

We used the same dilution from the same library to do emPCR amplification. All the condition is the same, the same lot kit and the same person to do this experiment. These interval experimental time is 3 days. We did twice but got different volume beads, one is 10% and another is almost 30%. How come the result is so different?? Does anyone has the same experience?? I'm so confused that the result is convincable?
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Old 08-18-2011, 11:01 AM   #2
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Are you using LVs, MVs, SVs or JR kits? Our JR kits tend to be consistent. But we see variation on the others - especially the MVs.
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Old 08-18-2011, 05:35 PM   #3
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JR means Junior? I used junior kit.
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Old 08-19-2011, 03:47 AM   #4
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Yes, Junior kits. I'm stumped as these tend to be the most consistent in our hands.
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Old 08-19-2011, 04:26 AM   #5
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Yes, we see these sorts of enrichment yield variations frequently. (Actually we use MV emPCR kits most of the time.)

How about room temp during emulsion formation? Seems like one major factor could be microreactor size, or microreactor size distribution. It seems like the viscosity of the emulsion oils would play a role here. Viscosity is often impacted by temperature. So if your lab is like mine you can see 4-5 oC temperature swings day to day. Maybe that is the cause? Just speculation, though.

Does anyone routinely check the size of microreactors with a microscope?

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Old 08-19-2011, 04:32 AM   #6
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Oh, another possible factor: how many times the emPCR kit gets freeze-thawed. I was surprised to see that Roche has the DNA capture beads stored frozen. A typical MV emPCR kit might be freeze-thawed 4 or 5 times. Each time all the beads are re-washed. Seems fairly traumatic to me.

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Old 08-22-2011, 06:52 PM   #7
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Is MV emPCR kits for FLX system? I can't change our protocol. My manager said how Roche trainer taught us then we did it. We wanna set up this system.
We always keep room temp at 22oC, we didn't change this condition, so there is no possible. And in my opinion emPCR kit is okay, emPCR kit wasn't freeze-thawed. Per kit is for one experiment. So we just thawed it for once.
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Old 08-22-2011, 11:37 PM   #8
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Yes, MV kits are used for FLX, usually when we divide the PTP in 4 or 8 regions.
In our lab we mostly use 1 kit per experiment too and we observe enrichment differences from time to time. We always titrate our libraries but scaling up from SV to MV is still a mystery to us. Luckily our Guess-O-meter works well.
For Sv kits when we thaw the kit for the first time we aliquot all the beads, and just wash the ones we are going to use for a given experiment.
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Old 08-23-2011, 03:49 AM   #9
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Quote:
Originally Posted by g861@hotmail.com View Post
[...]
We always keep room temp at 22oC, we didn't change this condition, so there is no possible. And in my opinion emPCR kit is okay, emPCR kit wasn't freeze-thawed. Per kit is for one experiment. So we just thawed it for once.
As implied earlier in the thread, we also see large variations of this sort from emPCR reactions. My guess is that after doing 100's of emPCRs you learn what the critical parameters are. Sadly the people doing 100's of emPCRs either don't read this forum at all or don't feel the need to post in response to questions of this sort.

I can only suggest some of the factors we try to lock down:

(1) Use low bind tips and tubes for all work with diluted libraries. Even low levels of binding by plastics will alter the concentration of your diluted libraries.

(2) Check the calibration on pippettes periodically by weighing water on an analytical scale.

(3) It would be nice to add some additional quality control points to the procedure. I think looking at the microreactors under a microscope might be useful. Also maybe running a little of the aqueous phase of the emPCR reaction on a bioanalyzer chip might help.

Anyone else have recommendations for making the emPCR protocol more "robust" (repeatable with similar results)?

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Old 08-23-2011, 11:25 PM   #10
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Quote:
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Yes, MV kits are used for FLX, usually when we divide the PTP in 4 or 8 regions.
Sorry, I don't understand you mean you separate PTP into 4 or 8 regions? I don't know about FLX- Titanium system, we only have Junior- Titanium machine. In junior, we can't divide PTP.
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Old 08-23-2011, 11:46 PM   #11
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Quote:
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(3) It would be nice to add some additional quality control points to the procedure. I think looking at the microreactors under a microscope might be useful. Also maybe running a little of the aqueous phase of the emPCR reaction on a bioanalyzer chip might help.
How to look at the microreactors under a microscope?
Another, emPCR is for DNA amplification in beads. DNA is already captured by beads, is that any effect in bioanalyzer running?
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Old 08-24-2011, 06:28 AM   #12
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Quote:
Originally Posted by g861@hotmail.com View Post
Sorry, I don't understand you mean you separate PTP into 4 or 8 regions? I don't know about FLX- Titanium system, we only have Junior- Titanium machine. In junior, we can't divide PTP.
Oh and I am not familiar with Junior at all. With FLX we can divide the plate in 2, 4, 8 and 16 regions using a rubber gasket. So depending on how many regions we divide the plate, we can choose in between different emulsion oil presentations: LV (large volume), MV (medium volume) and SV (small volume). In our lab we usually use LV for 2 regions, MV for 4 and SV for 8 or 16.
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Old 08-25-2011, 12:49 PM   #13
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I've seen this from time to time as well. Just recently I had a couple of libraries I titrated using an SV kit then scaled up to an MV kit. With the amount of DNA that gave me ~10% enrichment at the SV scale, I got only ~2-3% with the MV kit. I had to redo it to get enough beads. This time, I remade my dilution from the library stock and used the same amount as before. The result was the same ~10% enrichment I should have gotten the first time. It seems that an extra freeze-thaw cycle of 10^7 concentration library reduced its titer. I can't say for sure that's what happened, but that's they only way I can explain it at the moment.
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Old 08-26-2011, 03:38 AM   #14
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Quote:
Originally Posted by g861@hotmail.com View Post
How to look at the microreactors under a microscope?
Just pippette a little on to a microscope slide, put a cover slip over it (if necessary), and look under the microscope. Take a picture, if possible. Then, over time, you might see differences that correlate with emPCR results.
Quote:
Originally Posted by g861@hotmail.com View Post
Another, emPCR is for DNA amplification in beads. DNA is already captured by beads, is that any effect in bioanalyzer running?
You don't want to run the beads, just a ul of the aqueous solution from which you recover the beads. I guess only a tiny percentage of the total PCR reaction products are captured by the DNA capture beads.

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Old 08-26-2011, 06:28 AM   #15
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Quote:
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You don't want to run the beads, just a ul of the aqueous solution from which you recover the beads. I guess only a tiny percentage of the total PCR reaction products are captured by the DNA capture beads.

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One of the PCR primers for emPCR is attached to the bead, so one strand of the PCR products is always attached to the bead. Some of the other strand will be in the aqueous phase, but recovering that would be difficult, since it will be in a large volume of isopropanol mixed with the emulsion oil. If you want to run a Bioanalyzer trace of the emPCR products, your best bet would be to use the supernatant from the melt solution step after breaking the emulsions. It would probably be a good idea to EtOH precipitate it first to get rid of the NaOH.
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Old 08-26-2011, 07:12 AM   #16
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I am pretty sure there is a considerable amount of PCR product in the aqueous phase (not attached to the beads). I think some unanchored B primer is included in the PCR primer cocktail. Also, if all the amplicons end up attached to the beads, I don't see the reason to be so vigilant about amplicon contamination of the lab during emulsion breaking.

I agree the isopropanol used for breaking would be an issue though.

How about taking an aliquot of the beads (prior to melting with NaOH), heat denature to release the second strands, pull down the beads with a magnet and pull off the second strands and run them on an RNA chip? That way no need to do the ethanol precipitation.

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Old 08-26-2011, 07:46 AM   #17
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You may be right about there being some B primer in the amplification primer mix. If so, I wasn't aware of it. Either way, there will be some PCR product in the aqueous phase (single stranded if no unanchored B primer, double stranded if there is unanchored B primer). Beside the aqueous phase, the supernatant from the melt step is a also a contamination risk. All of those second strands that are melted off the beads (and whatever PCR product is in the aqueous phase) are at a high concentration and a fantastic template for emPCR.

As for your suggestion of heat denaturing the second strands, I suppose you could do that. You would have to get them pretty hot (94C) to melt them off, but it should work. I'm not sure what you would gain by doing so, but it shouldn't hurt.
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Old 08-26-2011, 10:05 AM   #18
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QC step. If run these 2nd strand on a RNA Pico chip and see lots of low molecular weight stuff you would suspect adapter dimers in your library. You also might get a general idea of how heavily templated the beads are over all.

I am just looking for ways to shine a light into this darkened room of the protocol.
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