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Primer Annealing Temperatures


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#16 Inbox

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Posted 26 August 2012 - 08:09 AM

I did a PCR once with primers one at 46 and the other one at 71 (long story, but I couldn't design them better no matter what I did, and I struggled quite a lot). Worked perfectly....but I'm the one with a too complicated PCR program Posted Image

Andreea

did you got some amplification ? How did you do that?

#17 ascacioc

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Posted 26 August 2012 - 03:49 PM

see my previous posts, after I said that I did that I also posted a protocol I use for Phusion PCR.

Andreea

#18 Trof

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Posted 04 October 2012 - 03:00 AM

Open source is a nice thing and surely if someones digs out the right composition by trying and googling good for him, on the other hand I have no problem with paying company for a good product they developed :)

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#19 brassmonkey

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Posted 03 March 2014 - 02:31 PM

I know this isn't at all timely, but Andreea mentioned SB buffer.  The same authors (Brody and Kern) later published on LB buffer (Lithium instead of sodium).  Now I run my gels for 10 min at 300V in Owl chambers rated to 150V and everything is great.  LB definitely out-performs SB.  Also a phusion lover, I'm going to try your touchdown protocol.  I've become less-enamored with touchdowns over the last couple of years, but I'll entertain your protocol.



#20 Trof

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Posted 14 March 2014 - 01:42 PM

I know this isn't at all timely, but Andreea mentioned SB buffer.  The same authors (Brody and Kern) later published on LB buffer (Lithium instead of sodium).  Now I run my gels for 10 min at 300V in Owl chambers rated to 150V and everything is great.  LB definitely out-performs SB.  Also a phusion lover, I'm going to try your touchdown protocol.  I've become less-enamored with touchdowns over the last couple of years, but I'll entertain your protocol.

What about the resolution of LB gel of higher framents? Or it only outperformes SB bufers in it pros but doesn't compensate much for cons?


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#21 brassmonkey

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Posted 14 March 2014 - 02:07 PM

How large of fragment do you need?  I get very sharp resolution of my ladder (KAPA ladder, 100bp-8000bp) after 10 min.  I should really measure the electrode-electrode distance so I could provide you V/cm which is actually useful.  Uploading images is not an obvious function of this forum, or I would proudly show you a picture.  I bet you could do PFGE with LB and get results in less time.



#22 Trof

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Posted 19 March 2014 - 04:54 AM

We have different fragment lengths. 

Generaly bands bigger than 3kb were not distiniguished on my ladder (that was with 2% agarose gel, but results were similar with 1% gel - which we generaly use)

 

12-10-16 SB-3.jpg

 

I run this quite long to see where the resolution would go, I usually only use SB buffer for very small fragments. 2-log ladder.

 

(PS: attaching images is easy, but you have to use More reply options button, not the Quick Reply, then you select the image, press Attach This File, and then you click on Add to post at the position you like)


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#23 brassmonkey

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Posted 19 March 2014 - 09:23 AM

OK, here's a gel image.  The ladder is KAPA Express.  Bands are 100, 200, 400, 800, 1600, 4000, 8000bp.

 

I recall the first time I tried SB I had a similar result as you show.  Now that my protocol is nailed down, everything is awesome.  My undergrads make the buffer it's so easy.

 

20X LB recipe: Add 8.39g LiOH pellets to 800mL H2O.  Then add 36g boric acid.  Bring volume to 1L.  No need to autoclave electrophoresis buffer.  Dilute to 1X for routine use (I use a 20L carboy, so dump in the 20X, fill to the top with RO water, good for a couple of months).

 

Also attached is my old SB protocol with references for each buffer, cost for each buffer.  Brody and Kern mentioned in 2 papers I am aware of that they estimate a loss of about $30 million to researchers due to adherence to the use of TAE/TBE for so long.

Attached Thumbnails

  • 2014-02-26 SSU troubleshooting gel2.jpg

Attached Files



#24 brassmonkey

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Posted 19 March 2014 - 09:30 AM

Electrophoresis conditions for that gel are as follows:

 

2uL each PCR product, 1.5uL KAPA Express Ladder

1X LB buffer

14V/cm (300V power supply, 21.5cm electrode-electrode distance)

150mA

12min run time

1% agarose (cheap Fisher agarose)

Gel dimensions 14cm long, 12cm wide

Total migration distance for each comb is about 2.5cm



#25 brassmonkey

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Posted 19 March 2014 - 09:58 AM

For gel imaging, make sure to get a good focus.  I use a software called Carestream (stupid name, decent software), though I have used software by AlphaInnotech, USB, Kodak, and others.  I keep the camera aperture fully open to get my focus (250ms exposure), then increase exposure time to 2s for image capture.  I know cameras and transilluminators (I use Invitrogen blue imager with SYBRsafe at 1/3rd concentration - 3.5uL per 85mL gel) can vary as well, but play around a bit and you can probably improve your results.  

 

One last thing to consider is the thickness of your gel.  Thinner gels give better results in my experience.  Electrophoresis apparatus manufacturers seem to be in cahoots with agarose suppliers to ensure we consume as much reagent as possible.  Place your gel tray into the casting stand with the comb in place until there is enough depth that the teeth are submerged "enough."  By enough, I mean, are they barely underwater?  If so, your DNA won't stay within the gel.  A few mm is all it takes.  Once the right depth is determined, use a graduated cylinder to measure the volume required to achieve this depth and adjust your protocol accordingly.  This will save you on agarose as well as buffer.  For some casting trays, using a leveling bubble when setting the tray is important so that your gel is level and uniform, or else samples can migrate off the gel surface if the gel is sloped too much.






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