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Search: Posts Made By: mdalessio
Forum: Illumina/Solexa 11-01-2013, 06:34 AM
Replies: 9
Views: 5,289
Posted By mdalessio
Thanks for all of the comments. I agree that...

Thanks for all of the comments.

I agree that the information is very fragmented. I think I am going to try to load ~8 pM and try again.
Forum: Sample Prep / Library Generation 10-31-2013, 01:44 PM
Replies: 2
Views: 2,788
Posted By mdalessio
DNA ng to pM help

Sorry to bother everyone, but I am having problems with my conversion.

So, my sample has 0.05 ng/ul

This is the same as 0.05 kg/L

0.05 kg/L x 1 mol / 330 kg [based on 1 kb length ssDNA]
...
Forum: Bioinformatics 10-28-2013, 02:04 PM
Replies: 4
Views: 957
Posted By mdalessio
I may not 100% understand what you are doing, but...

I may not 100% understand what you are doing, but you can just edit FASTA in a text editor like gedit and just save it
Forum: Illumina/Solexa 10-28-2013, 01:35 PM
Replies: 9
Views: 5,289
Posted By mdalessio
I guess inconsistencies isn't the right word, I...

I guess inconsistencies isn't the right word, I was more referring to what you are saying about the perceived "simplicity". In their protocol they don't even explain cluster density at all. For the...
Forum: Illumina/Solexa 10-28-2013, 12:10 PM
Replies: 9
Views: 5,289
Posted By mdalessio
Yes, this is my first run. I diluted phiX to...

Yes, this is my first run. I diluted phiX to 12.5 pM, and then added 5 ul of that, so far I am getting lower than ideal cluster density (~260K) but so far so good.

Makes sense about the...
Forum: Illumina/Solexa 10-28-2013, 09:11 AM
Replies: 9
Views: 5,289
Posted By mdalessio
Loading Nextera XT in to MiSeq

Hello.

Just went to load my diluted DAL sample, but the MiSeq user manual wants you to add 10 ul of phiX DNA to 990 ul diluted sample.

The Nextera XT kit outputs 600 ul of diluted sample.
...
Forum: Illumina/Solexa 10-24-2013, 08:13 AM
Replies: 6
Views: 3,009
Posted By mdalessio
Question about pooling calculations (Nextera XT)

I just used a Qubit to quantify all of my 24 samples. I am getting concentrations between 0.2 and 7 ng/ul for each sample. The issue is that I do not have access to a bioanalyzer, so should I...
Forum: Illumina/Solexa 10-21-2013, 06:51 AM
Replies: 18
Views: 5,072
Posted By mdalessio
I am a first time MiSeq user, so I am a bit...

I am a first time MiSeq user, so I am a bit confused. My understanding was that the MiSeq does its own clustering once you load the single sample well. Am I wrong?
Forum: Illumina/Solexa 10-20-2013, 10:46 AM
Replies: 2
Views: 2,111
Posted By mdalessio
How to accurately pipette genomic DNA

Hey everyone,

How do you pipette genomic DNA samples accurately? I have tried letting them sit at 65 C to encourage their equal distribution in the sample. For the XT kit it only wants 1 ng of...
Forum: Illumina/Solexa 10-19-2013, 06:27 AM
Replies: 18
Views: 5,072
Posted By mdalessio
Ah sorry, I meant NPM. I was flustered/upset...

Ah sorry, I meant NPM. I was flustered/upset about my obvious stupidity.

I did end up talking to Illumina, and all will be okay
Forum: Illumina/Solexa 10-18-2013, 12:27 PM
Replies: 18
Views: 5,072
Posted By mdalessio
Follow up due to my stupidity: If I am out...

Follow up due to my stupidity:

If I am out of the Amplicon Tagmentation Mix, do you think I could just use a regular DNAP instead? The transposome puts on the recognition sequence, and the PCR...
Forum: Illumina/Solexa 10-18-2013, 12:21 PM
Replies: 18
Views: 5,072
Posted By mdalessio
oh my goodness. I just had to do the biggest...

oh my goodness.

I just had to do the biggest face palm. I was trying to use the Nextera protocol, for the XT kit. I just want to slap myself. How could I be so stupid?!
Forum: Illumina/Solexa 10-18-2013, 08:50 AM
Replies: 18
Views: 5,072
Posted By mdalessio
Does anybody know when they switched the XT...

Does anybody know when they switched the XT protocol?
Forum: Illumina/Solexa 10-18-2013, 06:46 AM
Replies: 18
Views: 5,072
Posted By mdalessio
Thanks everyone. I am thinking that I will...

Thanks everyone.

I am thinking that I will qubit and bioanalyze after the PCR clean up, but if things look good, I think I will attempt the bead normalization, and check again afterwards to see...
Forum: Illumina/Solexa 10-16-2013, 10:07 AM
Replies: 18
Views: 5,072
Posted By mdalessio
Thanks. I have 24 samples to pool, so I am...

Thanks.

I have 24 samples to pool, so I am very tempted to use the beads. We also don't have our own bioanalyzer, so I have to use another labs - which I can, but I just feel bad.

I will look...
Forum: Sample Prep / Library Generation 10-16-2013, 06:23 AM
Replies: 14
Views: 12,282
Posted By mdalessio
Has someone already optimized the input DNA for...

Has someone already optimized the input DNA for genomic libraries, or is this something everyone should optimize themselves?
Forum: Illumina/Solexa 10-16-2013, 06:07 AM
Replies: 18
Views: 5,072
Posted By mdalessio
Nextera XT sample prep from genomic samples - question about QC along the way

Hello,

The newest version of the XT kit does not include the suggestions to do QC along the way anywhere - which makes me very nervous.

The older protocol had you check via a BioAnalyzer chip...
Forum: Illumina/Solexa 10-16-2013, 06:05 AM
Replies: 4
Views: 3,156
Posted By mdalessio
The newer Nextera XT kit contains a bead step for...

The newer Nextera XT kit contains a bead step for library normalization, but it does not replace the need for the AMpure XP beads after PCR - as the kit bead step does not come until after that.
Forum: Illumina/Solexa 10-16-2013, 06:04 AM
Replies: 11
Views: 8,578
Posted By mdalessio
Re. column purification for clean ups I did...

Re. column purification for clean ups

I did a column purification for clean up after the tagmentation and then did the PCR. While the PCR did work, I believe my yield was quite affected by the...
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