SequalPrep Normalisation kit low DNA concentration

katherine.wong · 02-09-2017, 05:47 AM

Has anybody had problems with getting lower than expected DNA concentrations for your normalised amplicons using the SequalPrep Normalisation kit?

According to their protocol, the expected DNA concentration is 1-2ng/ul when using 20ul elution volume. My group is consistently getting below this (0.2-0.8ng/ul). As suggested by the protocol, the starting material we used is >250 ng amplicon per well.

Thanks,
Katherine

laure · 02-13-2017, 06:54 AM

Hi Katherine,

I have exactly the same problem with this kit. I don't have found the solution yet but I can tell you that I have the same yield of you (mean at 0.3 ng/µL).
I have checked if my amplification is good and it's the case (25-50 ng/µL of PCR product obtained after amplification)
I have tested the normalization with exactly 250 ng of amplicons and more than 250 ng but I always a low concentration after normalization (<1 ng/µL).

I have also a very high variations between my samples after normalization (I can have amplicon A with a concentration at 0.1 ng/µL, amplicon B with 0.5 ng/µL, amplicon C with 0.7 ng/µL, ...) Do you see the same thing?
I hope someone can help us.

Thanks,

Laure

katherine.wong · 02-13-2017, 07:06 AM

Hi Laure,

Thanks for your reply. It's good to know we aren't the only ones experiencing this.

I find although I am getting low yields, the amplicons are fairly normalised (amplicon A at 0.29 ng/ul, amplicon B at 0.30 ng/ul, amplicon C at 0.25 ng/ul etc).

My colleague is speaking with Invitrogen's tech support so hopefully, they can help. Apparently, they have not heard of any problems from their other users.

Can I ask what is the lot number of the kit you are using? Mine is 1780013.

Thanks,
Katherine

katherine.wong · 02-20-2017, 06:15 AM

Hi again Laure,

My colleague has been speaking with Tech Support and they are replacing our kit.

They also suggest that you write in if you are having problems. Djamal is the person we have speaking with. They may replace yours or if it's a common problem, they may enquire further.

I hope this helps,
Katherine

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02-09-2017, 05:47 AM	#1
katherine.wong Junior Member Location: London Join Date: Nov 2016 Posts: 3	SequalPrep Normalisation kit low DNA concentration Has anybody had problems with getting lower than expected DNA concentrations for your normalised amplicons using the SequalPrep Normalisation kit? According to their protocol, the expected DNA concentration is 1-2ng/ul when using 20ul elution volume. My group is consistently getting below this (0.2-0.8ng/ul). As suggested by the protocol, the starting material we used is >250 ng amplicon per well. Thanks, Katherine

02-13-2017, 06:54 AM	#2
laure Junior Member Location: lyon Join Date: Jun 2016 Posts: 1	SequalPrep Normalisation kit low DNA concentration Reply to Thread Hi Katherine, I have exactly the same problem with this kit. I don't have found the solution yet but I can tell you that I have the same yield of you (mean at 0.3 ng/µL). I have checked if my amplification is good and it's the case (25-50 ng/µL of PCR product obtained after amplification) I have tested the normalization with exactly 250 ng of amplicons and more than 250 ng but I always a low concentration after normalization (<1 ng/µL). I have also a very high variations between my samples after normalization (I can have amplicon A with a concentration at 0.1 ng/µL, amplicon B with 0.5 ng/µL, amplicon C with 0.7 ng/µL, ...) Do you see the same thing? I hope someone can help us. Thanks, Laure

02-13-2017, 07:06 AM	#3
katherine.wong Junior Member Location: London Join Date: Nov 2016 Posts: 3	Hi Laure, Thanks for your reply. It's good to know we aren't the only ones experiencing this. I find although I am getting low yields, the amplicons are fairly normalised (amplicon A at 0.29 ng/ul, amplicon B at 0.30 ng/ul, amplicon C at 0.25 ng/ul etc). My colleague is speaking with Invitrogen's tech support so hopefully, they can help. Apparently, they have not heard of any problems from their other users. Can I ask what is the lot number of the kit you are using? Mine is 1780013. Thanks, Katherine

02-20-2017, 06:15 AM	#4
katherine.wong Junior Member Location: London Join Date: Nov 2016 Posts: 3	Hi again Laure, My colleague has been speaking with Tech Support and they are replacing our kit. They also suggest that you write in if you are having problems. Djamal is the person we have speaking with. They may replace yours or if it's a common problem, they may enquire further. I hope this helps, Katherine