TITLE: Ligation (Instructor Protocol)
AUTHORS: Brian    Teague

[DESCRIPTION]
This is the instructor protocol for the student Ligation protocol.


The abstract for the student protocol explains the basics. I pre-digest the L2-01 backbone for my students, but I generally do not gel-purify it. I find that the ligation is generally pretty robust, with three caveats:

Because I don't gel-purify, we need to bias the ligation towards ligating the annealed oligos (not the GFP that was digested out). To do so, I dilute the backbone to 10 fmol/ul and instruct the students to dilute their annealed oligos to 200 fmol/ul (which is 200 nanomolar (nM)).
Ligase is EXPENSIVE, and this kind of ligation does not need much -- so I dilute 1:5 into small tubes. Thus, if a beginning student screws something up (throws the tube away, or contaminates it, etc), the expensive master reagent tube isn't lost.
The ligase buffer contains ATP, which is heat-labile. (When a ligation fails, it's often because the buffer is bad, not the enzyme!) Thawing and re-freezing is not good for it. So, I aliquot the ligase buffer into single-use 5 ul aliquots in PCR tubes.


[STEPS]
SECTION: Grow &amp; miniprep L2-01
1. At least 48 hours before the lab, strike out the L2-01 E. coli strain from a frozen stock on an LB+Kan plate.

 

SECTION: Grow &amp; miniprep L2-01
2. At least 24 hours before the lab: pick a colony of L2-01 into 5 mL LB+Kan liquid media. Grow in a round-bottomed test-tube overnight on a shaker,   

 

 


SECTION: Grow &amp; miniprep L2-01
3. Miniprep the culture using the   or another suitable miniprep kit.

SECTION: Grow &amp; miniprep L2-01
4. Analyze the eluate on a Nanodrop for DNA concentration and purity.

 

SECTION: Linearize L2-01
5. We're going to linearize the ENTIRE miniprep. To a 200 ul PCR tube, add:
 50 µL miniprep
38 µL  
10 µL  
2 µL  

Flick several times to mix well, then pulse down in a microcentrifuge. 

SECTION: Linearize L2-01
6. Incubate 37 °C  for 180 min , then inactivate the reaction at 65 °C for 20 min

SECTION: Linearize L2-01
8. Optional: dilute with   to a final concentration of 10 fmol/µl.

SECTION: Aliquot / dilute reagents
9. Aliquot 1 mL of   per 4 students. Store at Room temperature.

SECTION: Aliquot / dilute reagents
10. Aliquot   into 5 µL single-use aliquots in 200 ul PCR strip tubes. Store at -20 °C  .

 


SECTION: Aliquot / dilute reagents
11. Dilute   1:5 in   . I make tubes containing 2 µL of ligase and 8 µL of buffer, one per 4 students. Store at -20 °C .

SECTION: Instructor Tips &amp; Common Student Errors
12. Instructor Tips
The first step of the student protocol requires another dilution. Again, I'm hands-off for this one. A common student question is "what is the concentration of my annealed oligos?" and I suggest that they might be able to figure it out by reviewing the annealing protocol.
I usually instruct students to run this protocol and the URA3 PCR protocol at the same time: set up the ligation, then while the ligation is incubating, set up their PCR.
SECTION: Instructor Tips &amp; Common Student Errors
13. Common Student Errors
Omitting the dilution step (particularly if they need to retry the protocol.)
Small-volume pipetting.
SECTION: Linearize L2-01
7. Optional: gel-purify the backbone. Run the entire miniprep on a preparative agarose gel, then use a kit such as the   to purify the larger band.