McClean: Yeast Recombinational Cloning

Michael T. Patel — Mon, 20 May 2013 17:17:40 GMT

Notes:

←Older revision		Revision as of 17:17, 20 May 2013
Line 41:		Line 41:
	* Optimized conditions for getting the plasmids back into bacteria (the hardest part so far)		* Optimized conditions for getting the plasmids back into bacteria (the hardest part so far)
	* Some examples of primer designs that have been successful		* Some examples of primer designs that have been successful
-	* If anyone does this with multiple fragments (ie, to also combine 1 or more fragments before they are inserted into the backbone) please add that to the ~~protocl~~	+	* If anyone does this with multiple fragments (ie, to also combine 1 or more fragments before they are inserted into the backbone) please add that to the protocol
		+
		+
		+	'''*[[User:Michael T. Patel\|Michael T. Patel]] 13:16, 20 May 2013 (EDT)''': Some observations/additions:
		+	* I typically transform DNA in a 2:1 ratio of PCR insert to digested vector. I usually eyeball the relative concentration by running equal volumes of each on a gel. I never use more than 10 μL of PCR product and 5 μL of vector.
		+	* Unless I'm going to use it for other purposes, I don't use the Qiagen purification kits to purify the digested vector. I just kill the enzyme according to NEB directions (usually just a heat-kill step)
		+	* I usually use yMM1146 (it has three common auxotrophies and I usually have it on hand) and any of the Sikorki-Hieter CEN plasmids (pMM5-8).
		+	* When I do this with multiple fragments, I maintain the above ratio with each fragment. So if I use 2 μL of vector, I'll use 4 μL of each fragment (assuming roughly equal concentration.)
		+	* Because we usually eyeball concentrations, it is probably helpful to plate at least 2 different concentrations of cells after transformation.
		+	* I also transform a control with only digested vector (no insert.) If your vector + insert plate has about 10 fold more colonies than the vector control, then your cloning likely worked.
		+

	Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.		Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.

McClean: Frogging a Serial Dilution

Michael T. Patel — Mon, 20 May 2013 16:37:17 GMT

New page

==Overview==
Frogging a serial dilution onto solid media is an effective way to distinguish and compare viability under certain growth conditions, etc. The serial dilution helps to eliminate the possibility that a relatively high cell concentration could be misinterpreted as healthy growth, for example.
==Materials==
* 96-well plate (flat bottom wells)
* Frogger
* Sterile petri dish about half-full of 95% ethanol
* Sterile petri dish about half-full of sterile water
* Multi-channel pipettor preferably with a range of 20 to 200 μL

==Protocol==
# Take your cells directly from culture (can be overnight or subculture) and load 200 μL of each sample into a well in the first column (left-most is conventional)of the 96 well plate.
# Fill the rest of the wells in the rows that you are using with 180 μL sterile water.
# Take the multi-channel pipettor, set it to 20 μL, pipette the cells in the first column of wells up and down a few times to suspend, take 20 μL from these wells and deposit it in the adjacent (to the right) column of cells. Pipette up and down to suspend.
# After you pipette to mix the cells, take 20 μL from this set of wells and do the same for the next. Continue until you reach the last column of wells on the plate.
# Sterilize the frogger by dipping the prongs into EtOH. After a quick shake insert the prongs into a Bunsen burner flame. Be CAREFUL not to burn yourself. Allow the frogger to cool slightly (1 minute). Alternatively, you can dip the frogger in sterile water for about 10 to 15 seconds to cool. Be sure to shake off the excess water. If you do not allow the frogger to cool enough, you will kill your cells and the frogging won't work well.
#Align the prongs of the frogger with the wells and then place the prongs into the wells. Wiggle the frogger within the well to ensure that the culture in each well is uniformly mixed. Stamp frogger firmly but gently onto an agar plate. Repeat this step to stamp additional plates.

==Notes==

==Contact==

*'''[[User:Michael T. Patel|Michael T. Patel]] 12:37, 20 May 2013 (EDT)''':or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].


[[Category:Protocol]]

OpenWetWare - Changes related to "Category:Protocol" [en]

McClean: Yeast Recombinational Cloning

McClean: Frogging a Serial Dilution