Stanford/BIOE44:Module 1:Day3

Introduction
You've already extracted plasmids from cells, digested them into fragments, and used the enzyme ligase to join these fragments with another unit of DNA containing a promoter sequence. Now you're faced with the challenge of getting that DNA back into a cell, so that you might observe the functional consequences of the genetic material you have assembled.

The take-up and expression of foreign DNA by cells is known as transformation. E.coli are not readily transformed as DNA does not freely move across their cell membrane. In order for transformation to take place, cells must be made competent. Competence is achieved by a variety of means. The means we will employ is electroporation, involving the use of an electrical shock. The shock temporarily creates holes in the E.coli cell membrane, allowing uptake of plasmid DNA.

Before you can shock the cells, you must prep them. This preparation involves centrifugation, removal of the supernatent, and washing of the cells to remove ions that interfere with the electrical shock.

In Class
Today you will combine your color generating insert with a "vector" containing an Isopropyl β-D-1-thiogalactopyranoside sensitive promoter. Together the two components will make up new plasmids, which you will need to transfer into cells. Today's experiments can be broken up into 4 major components: (1) Ligation (joining your insert with another piece of DNA, in this case: the promoter containing "vector"), (2) Preparation of cells for electroporation (getting the cell ready for "shock treatment"),(3) Transformation (shocking the cells so that they uptake your plasmids), and (4) Plating.

Ligation
Now that we have purified the gene cassettes, we will insert them into different vectors with inducible promoters. Next week we will characterize these composite parts. (FYI you guys will be making new composite parts that have never been made before... we may even submit these with our characterization data to the registry!)

You should have recorded answers to these questions from last lab. You will be using *R0011 You should have found in the registry and written down:
 * 1) What are the promoter names and what they induced by? :
 * 2) What are the concentrations of inducer relevant for induction?
 * 3) What plasmid are these parts on?
 * 4) What is the selection marker on the plasmid?
 * 5) The vectors with the inducible promoters you will get are precut, but at what sites were they cut?
 * 6) What is the size of cut vector?

Details of the ligation

We will be following this protocol taken DNA ligation. You need to determine what your ligation reaction components and volumes should be. This ligation calculator will be helpful.

Helpful hints:
 * 1) Don't forget to set up a ligation control. What would be a good control?
 * 2) Use a PCR tube.
 * 3) Set up a program on the PCR block that will incubate your ligation at room temperature for 30 minutes and then heat inactivate it at 65C for 10 minutes. (TAs will demonstrate how to write a program on the PCR block).

Procedure

Ratios for 10&mu;L Ligation Mix
Larger ligation mixes are also commonly used, (the ratio is the important thing here)
 * 1.0 &mu;L 10X T4 ligase buffer
 * 6:1 molar ratio of insert to vector (~10ng vector)
 * Add (8.5 - vector and insert volume)&mu;l ddH2O
 * 0.5 &mu;L T4 Ligase

Calculating Insert Amount
$${\rm Insert\ Mass\ in\ ng} = 6\times\left[\frac\right]\times{\rm Vector\ Mass\ in\ ng} $$

'''The insert to vector molar ratio can have a significant effect on the outcome of a ligation and subsequent transformation step. Molar ratios can vary from a 1:1 insert to vector molar ratio to 10:1. It may be necessary to try several ratios in parallel for best results.'''

Method

 * 1) Add appropriate amount of deionized H2O to sterile 0.6 mL tube
 * 2) Add 1 &mu;L ligation buffer to the tube. Vortex buffer before pipetting to ensure that it is well-mixed. Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation.
 * 3) Add appropriate amount of insert to the tube.
 * 4) Add appropriate amount of vector to the tube.
 * 5) Add 0.5 &mu;L ligase. Vortex ligase before pipetting to ensure that it is well-mixed. Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip.  To ensure you add only 0.5 &mu;L, just touch your tip to the surface of the liquid when pipetting.
 * 6) Let the 10 &mu;L solution sit at 22.5&deg;C for 30 mins
 * 7) Denature the ligase at 65&deg;C for 10min. (This is important since intact ligase will interfere with transformation in the next step of the experiment).

Electrocompetent Cell Prep
The first two steps of the protocol have been done for you.
 * 1) Pick an isolated colony from an LB plate and grow overnight in 3–5 ml of LB at 37C.
 * 2) Next morning, add 0.5 ml of the culture to 25 ml of LB in a 250-ml flask and grow at 37C to an OD600 of 0.50–0.60.
 * 3) Transfer the culture to a 50-ml Falcon tube and spin at 6,000g in prechilled rotor for 10 min at 4C.
 * 4) Wash the cell pellet with 20 ml of ice-cold H2O then centrifuge again as above.
 * 5) Resuspend the pellet in 1 ml of H2O and transfer to a chilled 1.5-ml tube. Spin at 10,000g for 30 seconds at 4C.
 * 6) Wash the cells again with 1 ml of ice cold H2O and centrifuge as above.
 * 7) Repeat the above wash and spin step.
 * 8) Resuspend the cell pellet in H2O in a final volume of 100μl and keep on ice.

Transformation via Electroporation
This procedure was adapted from a a full protocol by Knight

Procedure

 * 1) You should prepare a bucket of ice. (Chill electroporation cuvettes, DNA samples and tubes on ice through out the electroporation)
 * 2) Aloquot cells into pre-chilled 0.6mL tubes.
 * 3) Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes) or 2.5 kV (2mm cuvettes).
 * 4) Dial a P2 pipetman to 2&mu;L depending on the salt content of your DNA sample and.
 * 5) Dial a P200 pipetman to 50&mu;L or whatever volume of electrocompetent cells you want to use.
 * 6) Dial a P1000 pipetman to 950&mu;L and be ready to pipet in SOC (this is nutrient mixture including glucose that the cells will need to recover from electroporation).
 * 7) Pipet 1-2&mu;L of DNA sample and add to electrocompetent cells. Swirl tip around gently in cells to mix DNA and cells.  Do not pipet up and down.
 * 8) Place cells back on ice to ensure they remain cold.
 * 9) Transfer cell-DNA mixture to cuvettes using P200 pipetman. Try not to handle cuvette base too much so that it stays cold.
 * 10) Tap the cuvette on the counter gently so that cells are at the bottom and to remove any air bubbles.
 * 11) Place in chamber of electroporator.
 * 12) Slide the chamber in so that the cuvette sits snugly between electrodes.
 * 13) Pulse the cells with a shock by pressing button on electroporator. (Wait for the click).
 * 14) Remove cuvette from the chamber and immediately add SOC . This step should be done as quickly as possible to prevent cells from dying off.
 * 15) Transfer SOC-cell mixture to chilled eppendorf tube.
 * 16) Chill sample on ice for 2 mins to permit the cells to recover.
 * 17) Transfer eppendorf tube to 37&deg;C incubator; shake to promote aeration. Incubate for 1 hr to permit expression of antibiotic resistance gene.

Next Step is plating....

=Plating=

Now will be a great time to practice your sterile technique, so get out your Bunsen burner. You will need a LB-agar plate with Ampicillin and some sterile glass beads.


 * 1) Pour approximately 15 glass beads on to your plate (Don't count just quickly transfer them to minimize contamination)
 * 2) Pipet 200&mu;L of cells onto the center of the plate and close the lid.
 * 3) Place the plate on the bench and rapidly move the plate across the bench top at 90 degree angles. Don't swirl the glass beads as you won't spread your cells evenly.
 * 4) Once you believe the cell media has been spread across the plate, hold the plate vertically, so that the beads rest on the bottom edge. Slightly open the plate allowing the glass bead to fall into a collection plate. Again, only open the plate slightly to minimize potential contamination.
 * 5) Incubate plate overnight at 37&deg;C.
 * 6) Keep the remaining SOC-cell mixture on the benchtop in case you have to plate again.