SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
low library concentration biochembug Illumina/Solexa 0 08-02-2012 07:13 AM
Help with denaturing low concentration Illumina PE Library TonyBrooks Sample Prep / Library Generation 3 09-27-2010 09:30 AM
Better to risk low concentration library or further amplification? krobison Sample Prep / Library Generation 3 08-12-2010 02:40 PM
Low concentration GA library Melanie Sample Prep / Library Generation 4 08-04-2010 02:34 PM
Anyone use low concentration for ChIP-seq prep? Audry888 Sample Prep / Library Generation 4 05-28-2010 12:37 PM

Reply
 
Thread Tools
Old 05-20-2013, 12:26 PM   #1
Noa
Member
 
Location: haifa israel

Join Date: Jun 2011
Posts: 60
Default Preparation of low concentration libraries for hybridization

Hi,
I know this has been covered by many threads (I've read them all) but am looking for a few clarifications.

(1) I understand Illumina recommendations are such that you dont want to go past max 0.05 N NaOH during the DNA denaturization steps. But the threads I have seen here all had a 0.1N final NaOH concentraion during denaturing. Is this intentional/a mistake/a change in Illumina recommendations?

(2) Do you prepare libraries with the hyb buffer right before hyb to cBot (we are preparing it in an institute separate from the genome facility running the samples) or can they be saved for a few days at -20C (similar to Illumina recommendations for phiX libraries?) Anyone have experience here?

(3) Does the concentration at which the libraries are when they undergo denaturization matter? (One thread- http://seqanswers.com/forums/showthread.php?t=4435 - commented what the concentration was during denaturing was and I wasnt sure whether it was important)

(4) would be super-happy if someone could give my protocol a quick run-through before I actually go and do it.... especially happy if someone can look at the different calculations in part 1 and see if they all work out right and if any is preferable over the other.

1. Combine the following in a microcentrifuge tube:
template DNA (XXXXX μl)
1 N NaOH (1 μl)
Tris-Cl 10 mM, pH 8.5 (up to 20 μl)
XXXXX is such that the final concentration of these 20uL is 60pM. So for example, if you have a 1.5nM library, or 1500pM, you want to dilute it 25 fold (1500/60) so you would take 0.8uL library (0.8uL into 20uL is a 25 fold dilution).
Just as another example, if you have 0.6nM library, to get to 60pM you want to dilute 10 fold, so you would take 2uL library into the NaOH denaturing reaction.
(Alternatively- you can take more library and then further dilute with hyb buffer, but I am not sure why we would want to do that. For example, you could take from a 1.5nM library 13.3uL, which would give you a 1nM concentration in the 20uL (1.5*13.3/20). This you would then dilute in hyb buffer to a final concentration of 10pM in 120uL for center to load onto the cBot.)
(Alternatively, you could take the 600pM library, take 4uL, add 1uL 0.25 N NaOH, add 1uL 0.25N HCl, which would give you a 400pM solution. Now you take 3 uL of this into 117 uL hyb buffer for a final concentration of 10pM.)
2. Vortex briefly to mix the template solution.
3. Centrifuge the template solution to 280 xg for one minute.
4. Incubate for five minutes at room temperature to denature the template into single strands.
5. Add 1uL 1N HCl to neutralize the NaOH. Vortex briefly and spin down.
6. Transfer 20 μl of denatured template to a tube containing 100 μl of pre-chilled HT1 (Hybridization Buffer) to a final concentration of 10pM for loading onto chip (our center usually uses 8-12pM, recommended we use 10pM for a start).
7. From this step, keep the denatured template DNA on ice until you are ready to proceed to hybridization by cBot.
Noa is offline   Reply With Quote
Old 05-20-2013, 01:05 PM   #2
kcchan
Senior Member
 
Location: USA

Join Date: Jul 2012
Posts: 182
Default

Please see my responses in Red.

Quote:
Originally Posted by Noa View Post
Hi,
I know this has been covered by many threads (I've read them all) but am looking for a few clarifications.

(1) I understand Illumina recommendations are such that you dont want to go past max 0.05 N NaOH during the DNA denaturization steps. But the threads I have seen here all had a 0.1N final NaOH concentraion during denaturing. Is this intentional/a mistake/a change in Illumina recommendations?
If you're following the MiSeq protocol, it uses 0.2N NaOH in equal volume with the library, so the denaturing conditions will be 0.1N. I don't believe that the cBot protocol has been changed and still uses 0.1N NaOH.

(2) Do you prepare libraries with the hyb buffer right before hyb to cBot (we are preparing it in an institute separate from the genome facility running the samples) or can they be saved for a few days at -20C (similar to Illumina recommendations for phiX libraries?) Anyone have experience here?
Keep your libraries at 2nM (or 10nM) in TE or Illumina RSB until ready for cBot. Diluted DNA does not store very well. If the concentrations are lower than 2nM don't mess with it at all.

(3) Does the concentration at which the libraries are when they undergo denaturization matter? (One thread- http://seqanswers.com/forums/showthread.php?t=4435 - commented what the concentration was during denaturing was and I wasnt sure whether it was important)
The library concentration does matter somewhat during denaturing, it's not as important as the final concentrations.

(4) would be super-happy if someone could give my protocol a quick run-through before I actually go and do it.... especially happy if someone can look at the different calculations in part 1 and see if they all work out right and if any is preferable over the other.

1. Combine the following in a microcentrifuge tube:
template DNA (XXXXX μl)
1 N NaOH (1 μl)
Tris-Cl 10 mM, pH 8.5 (up to 20 μl)
XXXXX is such that the final concentration of these 20uL is 60pM. So for example, if you have a 1.5nM library, or 1500pM, you want to dilute it 25 fold (1500/60) so you would take 0.8uL library (0.8uL into 20uL is a 25 fold dilution).
Just as another example, if you have 0.6nM library, to get to 60pM you want to dilute 10 fold, so you would take 2uL library into the NaOH denaturing reaction.
(Alternatively- you can take more library and then further dilute with hyb buffer, but I am not sure why we would want to do that. For example, you could take from a 1.5nM library 13.3uL, which would give you a 1nM concentration in the 20uL (1.5*13.3/20). This you would then dilute in hyb buffer to a final concentration of 10pM in 120uL for center to load onto the cBot.)
(Alternatively, you could take the 600pM library, take 4uL, add 1uL 0.25 N NaOH, add 1uL 0.25N HCl, which would give you a 400pM solution. Now you take 3 uL of this into 117 uL hyb buffer for a final concentration of 10pM.)
2. Vortex briefly to mix the template solution.
3. Centrifuge the template solution to 280 xg for one minute.
4. Incubate for five minutes at room temperature to denature the template into single strands.
5. Add 1uL 1N HCl to neutralize the NaOH. Vortex briefly and spin down.
6. Transfer 20 μl of denatured template to a tube containing 100 μl of pre-chilled HT1 (Hybridization Buffer) to a final concentration of 10pM for loading onto chip (our center usually uses 8-12pM, recommended we use 10pM for a start).
7. From this step, keep the denatured template DNA on ice until you are ready to proceed to hybridization by cBot.

I got lost trying to follow what you're saying here. But here's my procedure for preparing low concentration libraries for clustering. This method has worked for me with libraries as low as 200pM.
  1. Add 18uL of library
  2. Add 2uL of 1N NaOH
  3. Incubate 5 minutes at room temperature, then place on ice
  4. Dilute to desired clustering concentration in HT1
  5. Add 2uL of 1N HCl to normalize pH. You can also use lower concentrations of HCl or a buffer if you like. The goal is to lower the pH to more neutral levels.
  6. Add PhiX (optional)
kcchan is offline   Reply With Quote
Old 05-20-2013, 11:21 PM   #3
Noa
Member
 
Location: haifa israel

Join Date: Jun 2011
Posts: 60
Default

Thanks for taking the time to answer: Further clarifications/questions:

(a) regarding the NaOH concentration- I am using the HiSeq 2500 - their protocol says to mix 10uL 0.1N NaOH with 10uL library, which would give a final concentration of 0.05N. Is this different in MiSeq?

(b) my library concentrations are low (<1nM) so I won't dilute them at all before preparing for the cBot. What I meant here was, after denaturing and adding hyb buffer, do I nned to immediately load this on the cBot or will this keep for some time?

(c) What range should I try to have my library concentration during denaturing?

(d) Thanks for the protocol. So for example if you do this on a 200pM library, after the NaOH you get a concentration of 180pM. Then if I want to load 10pM onto the cBot I need to dilute this 18-fold, so I would take 6.7uL of my denatured library into 113.3uL hybridization buffer?

Thanks!
noa
Noa is offline   Reply With Quote
Old 05-21-2013, 04:55 AM   #4
TonyBrooks
Senior Member
 
Location: London

Join Date: Jun 2009
Posts: 298
Default

If you add HCl, you neutralise the pH, so concentration of NaOH doesn't become an issue.

My protocol:

XXuL of Library for a 200pM in 20uL final volume (eg if lib is 500pM, then add 8uL)
XXuL water (for above example add 8uL water)
2uL 2N NaOH
--------------------
Total volume = 18uL

Incubate at RT for 5 mins

Add 2uL 2N HCl

Final volume is 200pM in 20uL. Dilute with HT1 to required concentration
TonyBrooks is offline   Reply With Quote
Old 05-21-2013, 05:01 AM   #5
Noa
Member
 
Location: haifa israel

Join Date: Jun 2011
Posts: 60
Default

Are you also using MiSeq? Why is everyone using a final of 0.1N NaOH? The protocol I have from Illumina uses a final of 0.05N (see below):

1 Combine the following volumes of template DNA and 0.1 N NaOH in a
microcentrifuge tube:
2 nM template DNA (10 μl)
0.1 N NaOH (10 μl)
2 Vortex briefly to mix the template solution.
3 Centrifuge the template solution to 280 xg for one minute....

Thanks for the info!
Noa is offline   Reply With Quote
Old 05-22-2013, 06:37 AM   #6
microgirl123
Senior Member
 
Location: New England

Join Date: Jun 2012
Posts: 192
Default

Where are you getting your Illumina protocol from? All the versions I have seen call for you to dilute the NaOH to 0.2 N (200 ul 1 N + 800 ul H2O) then add an equal amount to your DNA - 0.1 N. This is for the MiSeq.
microgirl123 is offline   Reply With Quote
Old 05-22-2013, 10:58 AM   #7
Noa
Member
 
Location: haifa israel

Join Date: Jun 2011
Posts: 60
Default

the protocol is from our genome center- Illumina cBot user guide - (manual is Illumina part # 15006165 rev. K) from Oct 2012

(attached)

note it specifically says:

NOTE
If your application requires higher than a 20 pM final concentration of your library,
make sure that your concentration of NaOH is not higher than 0.05 N in the
denaturation solution and not more than 0.001 N (1 mM) in the final solution after
diluting with HT1.

1 Combine the following volumes of template DNA and 0.1 N NaOH in a
microcentrifuge tube:
• 2 nM template DNA (10 μl)
• 0.1 N NaOH (10 μl)
Attached Files
File Type: pdf cBot_UG_15006165_K_Public.pdf (3.24 MB, 27 views)
Noa is offline   Reply With Quote
Old 05-22-2013, 11:19 AM   #8
microgirl123
Senior Member
 
Location: New England

Join Date: Jun 2012
Posts: 192
Default

That's your problem! I think you're getting answers about the MiSeq and the HiSeq interchangeably. I think that the previous posts about using more concentrated NaOH, but neutralizing it after denaturation, should work. I would call or email Illumina tech support with your proposed method and see what they think.
microgirl123 is offline   Reply With Quote
Old 05-22-2013, 11:22 AM   #9
ECO
--Site Admin--
 
Location: SF Bay Area, CA, USA

Join Date: Oct 2007
Posts: 1,355
Default

Neutralizing the reaction in a small volume can lead to variability ( ostensibly by partial/reannealing of adapter regions), we've found that neutralizing with HCl after addition of HT1 dramatically reduces our variability.

...and I've seen absolutely no difference betwen 50mM and 100mM final NaOH in the denaturation. Unclear why ILMN changed it.
ECO is offline   Reply With Quote
Old 05-22-2013, 11:24 AM   #10
microgirl123
Senior Member
 
Location: New England

Join Date: Jun 2012
Posts: 192
Default

So if you added 2 ul of 1N NaOH for denaturation, you would do all the dilutions up to 600 ul for the MiSeq (or whatever you wanted) with HT1, then add 2 ul of 1N HCl?
microgirl123 is offline   Reply With Quote
Old 05-22-2013, 11:38 AM   #11
Noa
Member
 
Location: haifa israel

Join Date: Jun 2011
Posts: 60
Default

Has anyone ever used this protocol on a HiSeq (and not a MiSeq)?
Thanks!
Noa is offline   Reply With Quote
Old 05-22-2013, 12:28 PM   #12
snorberg
Junior Member
 
Location: San Diego, CA

Join Date: Jan 2013
Posts: 5
Default

You are correct, the cBOT protocol recommends 0.5N NaOH denaturation and the MiSeq protocol 0.1N NaOH.

Regardless of the concentration of NaOH used to denature libraries, 0.05N or 0.1N, the rule of thumb is that [NaOH] in your final hyb not exceed 1mM. If [NaOH] is >1mM, a buffer must be used to netrualize the NaOH, as others have suggested.
snorberg is offline   Reply With Quote
Old 05-22-2013, 01:16 PM   #13
Noa
Member
 
Location: haifa israel

Join Date: Jun 2011
Posts: 60
Default

So does anyone have a clue why you would use different final NaOH concentrations to denature the same DNA? The libraries should be the same in the end regardless of whether they go on MiSeq or HiSeq, no? The same NaOH would denature the DNA I would assume? Just curious....
Noa is offline   Reply With Quote
Old 05-22-2013, 03:20 PM   #14
kcchan
Senior Member
 
Location: USA

Join Date: Jul 2012
Posts: 182
Default

Illumina has been playing around with the denaturing protocol of the MiSeq quite a bit in the past year. The initial protocol was identical to the cBot. In revision B, they doubled the NaOH concentration for denaturing, but did nothing to reduce the final NaOH concentration. Now they're at revision G where they've changed the protocol again where you use 4nM of DNA at 5uL and 0.2N of NaOH at 5uL to dilute the libraries, thus bringing the final NaOH concentrations back down to where they started.
kcchan is offline   Reply With Quote
Old 05-02-2018, 09:47 AM   #15
cement_head
Senior Member
 
Location: Oxford, Ohio

Join Date: Mar 2012
Posts: 209
Question

Quote:
Originally Posted by kcchan View Post
Illumina has been playing around with the denaturing protocol of the MiSeq quite a bit in the past year. The initial protocol was identical to the cBot. In revision B, they doubled the NaOH concentration for denaturing, but did nothing to reduce the final NaOH concentration. Now they're at revision G where they've changed the protocol again where you use 4nM of DNA at 5uL and 0.2N of NaOH at 5uL to dilute the libraries, thus bringing the final NaOH concentrations back down to where they started.
I fail to understand why people add back HCl at the end (I understand neutralising the NaOH, and bringing the pH back to "normal"), but shouldn't this be accomplished largely by the dilution of the denatured library in the HT1 buffer? In other words, when did the "add HCl" step start getting introduced and is there any evidence that it helps? Thanks.
cement_head is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 01:30 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO