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Forum: Illumina/Solexa 07-04-2014, 04:42 AM
Replies: 18
Views: 8,254
Posted By Neiltje
Just to make sure: you only adjust the adaptor...

Just to make sure: you only adjust the adaptor concentration, never the concentration of the enzymes used in the different steps?
Forum: Illumina/Solexa 06-27-2014, 01:35 AM
Replies: 18
Views: 8,254
Posted By Neiltje
Well, both the kits from NEB and KAPA start with...

Well, both the kits from NEB and KAPA start with 1-5 g start DNA.
I'm planning on tracking my samples on the bioanalyzer after every step the first time I do this, so I can see what happens with my...
Forum: Illumina/Solexa 06-26-2014, 11:19 PM
Replies: 18
Views: 8,254
Posted By Neiltje
That's a clear answer, thanks :-).

That's a clear answer, thanks :-).
Forum: Illumina/Solexa 06-26-2014, 06:44 AM
Replies: 18
Views: 8,254
Posted By Neiltje
sounds great, do you use the recommended input...

sounds great, do you use the recommended input concentration of adaptors? seems like overkill if your input DNA is so much lower.
Forum: Illumina/Solexa 06-26-2014, 03:11 AM
Replies: 18
Views: 8,254
Posted By Neiltje
Well, my amplicons are blunt ended so I basically...

Well, my amplicons are blunt ended so I basically need the total kit. And I have +/- 90 preps. The only difference is the entry DNA, since they are amplicons, the concentration is a lot lower. On...
Forum: Illumina/Solexa 06-26-2014, 02:49 AM
Replies: 18
Views: 8,254
Posted By Neiltje
we are planning to use the NEBNext DNA library...

we are planning to use the NEBNext DNA library prep kit with the multiplexed oligo's. I need exactly 24 indices so I'm lucky :-).

Why are you using KAPA prep kit instead of NEB?
Forum: Illumina/Solexa 06-20-2014, 03:36 AM
Replies: 18
Views: 8,254
Posted By Neiltje
Ligating adaptors sounds pretty good. I found a...

Ligating adaptors sounds pretty good. I found a kit from New england biolabs with indexed primers. Problem is, these are primers, so single stranded? And my amplicon is double stranded. How do I get...
Forum: Illumina/Solexa 06-20-2014, 03:09 AM
Replies: 18
Views: 8,254
Posted By Neiltje
Wink well, this is exactly what I can't use for the...

well, this is exactly what I can't use for the reasons mentioned above ;)
Forum: Illumina/Solexa 06-20-2014, 01:48 AM
Replies: 18
Views: 8,254
Posted By Neiltje
How to add illumina adaptors to amplicons?

How can you add Illumina adaptors to amplicons of +/- 300 bp? The ampliconlength is to small to use the nextera method. I tried by PCR by adding (a part of) the adaptor sequence to my specific...
Forum: Illumina/Solexa 05-16-2014, 07:15 AM
Replies: 4
Views: 5,532
Posted By Neiltje
I am also trying this protocol but I'm running...

I am also trying this protocol but I'm running into problems.
My DNA is bisulfite treated (all non-methylated C's are converted to U) so I'm using an enzyme from KAPA that is especially designed for...
Forum: Illumina/Solexa 05-16-2014, 06:00 AM
Replies: 1
Views: 2,433
Posted By Neiltje
primer dimer problem in PCR using Illumina protocol

Hi!
I'm using the two step PCR Amplicon approach to to some targeted sequencing.
My PCR with my primers without adaptor sequences works just fine. But when I add adaptor sequences according to the...
Forum: RNA Sequencing 02-20-2014, 06:28 AM
Replies: 2
Views: 1,635
Posted By Neiltje
We decided not to use this because we have a...

We decided not to use this because we have a bioinformatics team. Thanks for your answer!
Forum: Illumina/Solexa 02-20-2014, 06:05 AM
Replies: 7
Views: 2,173
Posted By Neiltje
Ok, thank you :) I just discovered Illumina has...

Ok, thank you :)
I just discovered Illumina has a lot of online training video's. They also clarify a lot.
Forum: RNA Sequencing 02-20-2014, 05:48 AM
Replies: 2
Views: 1,635
Posted By Neiltje
small RNA seq question Miseq

Hi,

I'm new in sequencing, next week I'm going to prepare a small RNA library using NEBNExt Multiplex small RNA prep kit for Illumina from New England biolabs.
I'm watching some tutorials from...
Forum: Illumina/Solexa 02-20-2014, 03:43 AM
Replies: 7
Views: 2,173
Posted By Neiltje
So my thinking was right, 50 cycles for a...

So my thinking was right, 50 cycles for a fragment of +/- 147 bp, will not cover the sequence in the middle? 50 bp from the right and 50 bp from the left, still leaves 47 bp in the middle.


About...
Forum: Illumina/Solexa 02-20-2014, 03:33 AM
Replies: 7
Views: 2,173
Posted By Neiltje
Oh and another question. Why is sequencing of a...

Oh and another question.
Why is sequencing of a PCR fragment on a Miseq overkill?
Forum: Illumina/Solexa 02-20-2014, 03:24 AM
Replies: 7
Views: 2,173
Posted By Neiltje
Miseq questions from a newbie

Hi,

I'm new in the sequencing world (apart from standard courses in university).
I've prepared an RNAseq library using the Scriptseq protocol from Epicentre. Together with a co-worker we...
Forum: Sample Prep / Library Generation 02-04-2014, 09:58 PM
Replies: 5
Views: 1,989
Posted By Neiltje
I'm using the RiboZero plant leaf on young leafs...

I'm using the RiboZero plant leaf on young leafs of rice. I think it just doesn't work well on this material because the last time I tried, I also took a sample of Canola and that worked fine...
Forum: Sample Prep / Library Generation 02-03-2014, 10:03 PM
Replies: 5
Views: 1,989
Posted By Neiltje
According to their protocol the input should be...

According to their protocol the input should be between 1 and 5 g. I'm using standard 2.5 g so that cannot be an issue :(
Forum: Sample Prep / Library Generation 01-30-2014, 06:37 AM
Replies: 5
Views: 1,989
Posted By Neiltje
RiboZero won't work?

Hi,

I'm trying to make a library of RNA following the ScriptSeq protocol. Everything works just fine except the RiboZero depletion of rRNA. I'm using young leafs of rice. An input of 2.5 g of...
Forum: Sample Prep / Library Generation 01-30-2014, 06:25 AM
Replies: 4
Views: 2,221
Posted By Neiltje
I also succeeded in making libraries out of very...

I also succeeded in making libraries out of very little starting material.
I face another problem: The riboZero reaction is inconsistent and sometimes doesn't work at all.
These are my parameters:...
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