Week 5
I nearly finished the cloning process this week but ran into my first bout of trouble. No project would be fun without a challenge, though, which is exactly what I encountered.
Most of the week was spent diagnosing then attempting a solution-- whether it works remains to be seen when I return!
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On Monday, I ran a gel on the DNA that was sitting in the fridge over the weekend (if you haven't noticed, I do a lot of gel electrophoresis, as the DNA has to be verified as correct after every step of the FastCloning process). Oddly, nothing showed up in the gel. I checked everything about the gel setup itself (including the agarose solution, this time), but nothing was wrong, meaning the DNA was the culprit.
Dr. Chang suggested that I amplify the DNA-- maybe the issue was that there simply wasn't enough of it. I spent the rest of the afternoon preparing to do just that.
On Tuesday, I performed the PCR and then--surprise--ran a gel on the amplified DNA. Still nothing. What could be wrong?
I spent my free day meeting up on Wednesday with my faculty advisor, Mr. Cole.
On Thursday, Dr. Chang told me to forget the pure DNA and amplify the bacteria themselves, then try to run that. As I opened the fridge to retrieve some materials, the source of my predicament revealed itself: I had used the wrong set of DNA on Monday.
After a few minutes (hours, really) of internal facepalming, I correctly labeled all my materials and ran a gel on the DNA I was actually supposed to use.
Most of the fragments failed. One did succeed, though! So this time, I knew there was something genuinely wrong with the DNA itself and not simply my absentmindedness.
I then tried running a gel on the amplified bacteria that were supposed to be the backup plan. To my pleasant surprise, most of the DNA migrated to their expected positions!
All, that is, except one. Of course.
So on Friday, I retraced a few steps in the cloning procedure and restarted from a point when the now-incorrect DNA had been confirmed by gel electrophoresis. Call it a "checkpoint" of sorts.
That checkpoint was last Wednesday, when I first inoculated the E. coli and spread them on Petri dishes. I did that once again and left them in the incubator, where they currently lie.
Will I finally be able to move on to the final step of cloning? Or will our hero face yet another obstacle in his path? Find out next time onDragon Ball Z my blog!
Most of the week was spent diagnosing then attempting a solution-- whether it works remains to be seen when I return!
--------------------------------------------------------------------------------------------------------------------------
On Monday, I ran a gel on the DNA that was sitting in the fridge over the weekend (if you haven't noticed, I do a lot of gel electrophoresis, as the DNA has to be verified as correct after every step of the FastCloning process). Oddly, nothing showed up in the gel. I checked everything about the gel setup itself (including the agarose solution, this time), but nothing was wrong, meaning the DNA was the culprit.
Dr. Chang suggested that I amplify the DNA-- maybe the issue was that there simply wasn't enough of it. I spent the rest of the afternoon preparing to do just that.
On Tuesday, I performed the PCR and then--surprise--ran a gel on the amplified DNA. Still nothing. What could be wrong?
I spent my free day meeting up on Wednesday with my faculty advisor, Mr. Cole.
On Thursday, Dr. Chang told me to forget the pure DNA and amplify the bacteria themselves, then try to run that. As I opened the fridge to retrieve some materials, the source of my predicament revealed itself: I had used the wrong set of DNA on Monday.
After a few minutes (hours, really) of internal facepalming, I correctly labeled all my materials and ran a gel on the DNA I was actually supposed to use.
Most of the fragments failed. One did succeed, though! So this time, I knew there was something genuinely wrong with the DNA itself and not simply my absentmindedness.
I then tried running a gel on the amplified bacteria that were supposed to be the backup plan. To my pleasant surprise, most of the DNA migrated to their expected positions!
All, that is, except one. Of course.
So on Friday, I retraced a few steps in the cloning procedure and restarted from a point when the now-incorrect DNA had been confirmed by gel electrophoresis. Call it a "checkpoint" of sorts.
That checkpoint was last Wednesday, when I first inoculated the E. coli and spread them on Petri dishes. I did that once again and left them in the incubator, where they currently lie.
Will I finally be able to move on to the final step of cloning? Or will our hero face yet another obstacle in his path? Find out next time on
Don't you just love having technical difficulties? Hopefully it all works out!
ReplyDelete"I knew there was something genuinely wrong with the DNA itself and not simply my absentmindedness."
ReplyDeleteWould you happen to know why the DNA failed?
I wish I could tell you. The truth is, there are so many things that can go wrong (cross-contamination, air bubbles, improper mixing, etc.) that simply retrying is often a better course of action that trying to determine the precise error.
Delete