Hi,
I'm to the NGS world and I just started my experiments the other day. I'm using a custom designed SureSelect XT capture kit and sheared my DNA using a Covaris M220. The protocol uses an S220/E220 but I don't have access to those machine and thought that the M220 would be all right. The settings that was in the SureSelect XT doesn't apply as one of the settings can't go that high on the M220. So I just sheared my DNA using the M220 protocol for fragmenting DNA to 150 bp as the SureSelect protocol wants fragments that are between 150 - 200 bp. I analysed the fragments on a Bioanalyzer high sensitivity DNA chip and found that the fragments are around 200 bp in size (the peak is between 220 - 300 bp on the Bioanalyzer). How important is the sheared DNA size and would this affect my downstream experiments?
Also, for the first AmPure bead extraction, part of the protocol suggest drying the pellet on a 37C heat block for 5 mins and not to let the pellet dry excessively as it affects elution. Someone changed my setting to 99C and I didn't realise and I over-dried my pellets. My elution is quite low for my samples... how much of an affect will this have on my downstream experiments?
Thank you
I'm to the NGS world and I just started my experiments the other day. I'm using a custom designed SureSelect XT capture kit and sheared my DNA using a Covaris M220. The protocol uses an S220/E220 but I don't have access to those machine and thought that the M220 would be all right. The settings that was in the SureSelect XT doesn't apply as one of the settings can't go that high on the M220. So I just sheared my DNA using the M220 protocol for fragmenting DNA to 150 bp as the SureSelect protocol wants fragments that are between 150 - 200 bp. I analysed the fragments on a Bioanalyzer high sensitivity DNA chip and found that the fragments are around 200 bp in size (the peak is between 220 - 300 bp on the Bioanalyzer). How important is the sheared DNA size and would this affect my downstream experiments?
Also, for the first AmPure bead extraction, part of the protocol suggest drying the pellet on a 37C heat block for 5 mins and not to let the pellet dry excessively as it affects elution. Someone changed my setting to 99C and I didn't realise and I over-dried my pellets. My elution is quite low for my samples... how much of an affect will this have on my downstream experiments?
Thank you
Comment