Stanford/BIOE44:Module 1:Day3

M1: Day 3 - Electrocompetent Cell Prep and Transformation

Introduction

In Class

Today you will combine your color generating insert with a "vector" containing an Isopropyl β-D-1-thiogalactopyranoside sensitive promoter into a new plasmid. Then you will need to transfer your plasmid into a cell where you may be able to observe the functional consequences of the genetic material you have assembled.

To do this you will need complete 3 major tasks: (1) Ligation (joining your insert with another piece of DNA, in this case: you promoter containing part), (2) Preparation of cell for electroporation (preparing the cell for "shock treatment"), and (3) Transformation (getting a cell to uptake one of your plasmids).

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 Find these parts in the registry and write 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=

10μL Ligation Mix

Larger ligation mixes are also commonly used, (the ratio is what is important here)

• 1.0 μL 10X T4 ligase buffer
• 6:1 molar ratio of insert to vector (~10ng vector)
• Add (8.5 - vector and insert volume)μl ddH₂O
• 0.5 μL T4 Ligase

Calculating Insert Amount

[math]\displaystyle{ {\rm Insert\ Mass\ in\ ng} = 6\times\left[\frac{{\rm Insert\ Length\ in\ bp}}{{\rm Vector\ Length\ in\ bp}}\right]\times{\rm Vector\ Mass\ in\ ng} }[/math]

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 μL 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 μL 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 μL, just touch your tip to the surface of the liquid when pipetting.
6. Let the 10 μL solution sit at 22.5°C for 30 mins
7. Denature the ligase at 65°C for 10min

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.
9. Mix 0.5uL of your ligation reaction with 50μl of electrocompetent cells transfer into a 0.1-cm cuvette.
10. Introduce the DNA into the cells by electroporation (1.8 kV, 25 mF capacitance and 200 O resistance).
11. After electroporation, immediately add 1 ml of SOC and transfer to 1.5mL microcentrigure tube.
12. Incubate cells (with shaking or rotation) at 37C for at least 30minutes
13. Spin down cells for 2 min at 4000g in a microcentrifuge.
14. Resuspend the pellets in 200 μl of LB. Plate each aliquot of cells on a single LB plate containing the appropriate antibiotic. Grow for 12-16 hours (overnight) at 37C.