GE health-maxiprep - (Aug/20/2008 )
firstly，i pick one clone for inoculating into 2ml LB，37℃，250rpm for 10hr。then I take the ratio 1：1000 reinoculate into 250ml LB medium 37℃，250rpm for 12-14hr。
secondly,after I used the kit solutionI II III according to the kit protocol,I found the supernatant is not clear,its colour seems to yellow,it apparently contained something,so I centrifuge more times at bakerman 8000g 30min,but the result is the same!
I'll be appreciated for your troubleshooting!
-aaronah86-
QUOTE (aaronah86 @ Aug 20 2008, 05:20 PM)
firstly，i pick one clone for inoculating into 2ml LB，37℃，250rpm for 10hr。then I take the ratio 1：1000 reinoculate into 250ml LB medium 37℃，250rpm for 12-14hr。
secondly,after I used the kit solutionI II III according to the kit protocol,I found the supernatant is not clear,its colour seems to yellow,it apparently contained something,so I centrifuge more times at bakerman 8000g 30min,but the result is the same!
I'll be appreciated for your troubleshooting!
secondly,after I used the kit solutionI II III according to the kit protocol,I found the supernatant is not clear,its colour seems to yellow,it apparently contained something,so I centrifuge more times at bakerman 8000g 30min,but the result is the same!
I'll be appreciated for your troubleshooting!
Keep going!!!
All is well! 
When you have a concentrated solution of nucleic acids, it can go slightly yellow. As long as the supernatant is not cloudy, you've spun down the crud (membranes, proteins, general assorted junk). -swanny-
QUOTE (swanny @ Aug 21 2008, 08:28 AM)
QUOTE (aaronah86 @ Aug 20 2008, 05:20 PM)
firstly，i pick one clone for inoculating into 2ml LB，37℃，250rpm for 10hr。then I take the ratio 1：1000 reinoculate into 250ml LB medium 37℃，250rpm for 12-14hr。
secondly,after I used the kit solutionI II III according to the kit protocol,I found the supernatant is not clear,its colour seems to yellow,it apparently contained something,so I centrifuge more times at bakerman 8000g 30min,but the result is the same!
I'll be appreciated for your troubleshooting!
secondly,after I used the kit solutionI II III according to the kit protocol,I found the supernatant is not clear,its colour seems to yellow,it apparently contained something,so I centrifuge more times at bakerman 8000g 30min,but the result is the same!
I'll be appreciated for your troubleshooting!
Keep going!!!
All is well! When you have a concentrated solution of nucleic acids, it can go slightly yellow. As long as the supernatant is not cloudy, you've spun down the crud (membranes, proteins, general assorted junk).thanks！but I tried more than 6 times,the supernatant is still yellow but seemed clear.I went on but found that the amounts of the pellet is huge after isopropanol pecipitation. I use the MQ solute the pellet,quite a lot didn't solute. so I spun it down,there were still lots of crud!
-aaronah86-
QUOTE (aaronah86 @ Aug 24 2008, 03:50 PM)
QUOTE (swanny @ Aug 21 2008, 08:28 AM)
QUOTE (aaronah86 @ Aug 20 2008, 05:20 PM)
firstly，i pick one clone for inoculating into 2ml LB，37℃，250rpm for 10hr。then I take the ratio 1：1000 reinoculate into 250ml LB medium 37℃，250rpm for 12-14hr。
secondly,after I used the kit solutionI II III according to the kit protocol,I found the supernatant is not clear,its colour seems to yellow,it apparently contained something,so I centrifuge more times at bakerman 8000g 30min,but the result is the same!
I'll be appreciated for your troubleshooting!
secondly,after I used the kit solutionI II III according to the kit protocol,I found the supernatant is not clear,its colour seems to yellow,it apparently contained something,so I centrifuge more times at bakerman 8000g 30min,but the result is the same!
I'll be appreciated for your troubleshooting!
Keep going!!!
All is well! When you have a concentrated solution of nucleic acids, it can go slightly yellow. As long as the supernatant is not cloudy, you've spun down the crud (membranes, proteins, general assorted junk).thanks！but I tried more than 6 times,the supernatant is still yellow but seemed clear.I went on but found that the amounts of the pellet is huge after isopropanol pecipitation. I use the MQ solute the pellet,quite a lot didn't solute. so I spun it down,there were still lots of crud!
OK, my bad. I didn't look closely at the parameters of your spin. I don't think 8000 x g will be enough to spin out all of the junk, unless the GE protocol is significantly different to all the others I know. Try spinning 30 minutes at >20,000 x g.
-swanny-