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.
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.
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