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Cloning 

What is Molecular Cloning?

Cloning by Infusion method 

Using Micro-pippettes

Micropipettes are used for accurately transferring small volumes of liquid in the milliliter and microliter range. We will cover three basic sizes of these handy little instruments.

    The P - 1000 accurately delivers 200 to 1000 microliters.
    The P- 200 accurately delivers 30 to 200 microliters.
    The P-20 accurately delivers 2 to 20 microliters.

The liquid is drawn into, and dispensed from a disposable pipette tip - The disposable tip is changed between liquid transfers. Pre-rinse every new pipette tip with the liquid to be pipetted.

Despite the differences in micropipette shapes and sizes, they have the same basic parts:

    a plunger for drawing up and expelling liquids
    a dial for tuning the pipette to different volumes
    the display, and an ejector for launching tips towards wastebaskets.

The volume is set by turning the dial. When decreasing the volume, slowly turn the dial to reach the required volume. Be careful not to overshoot. When increasing the volume, pass the desired volume by one-third turn and then slowly decrease the volume to the final mark.

The display is read from top to bottom. For a P-1000, the volume is given in milliliters. The top position is the ones place, the middle is the tenths place, and the bottom is the hundredths place. In the P-200, the top position is the hundreds place, the middle is the tens place, and the bottom is the ones place. The top position in the P20 is the tens place, the middle is the ones place, and bottom is the tenths place.

Normal operations consist of the following steps:

Preparation: Hold the instrument in a nearly vertical position. Depress the plunger smoothly to the first stop.
Aspiration: mmerse the pipette tip in the liquid. Allow the plunger to move up smoothly to the rest position. Wait one second so that the liquid has time to move up into the tip.
Distribution: Place the pipette tip at an angel of 10 to 45 degrees against the inside wall of the receiving vessel. Depress the plunger smoothly to the first stop position.
Purge: Wait one second, then depress the plunger to the second stop position. This “blow-out” stroke removes any remaining sample from the tip. Remove the pipette tip by sliding it up against the vessel wall.
Home: Allow the plunger to move up to the rest position. After transferring your liquid, you can eject the tip and start anew.

To ensure maximum accuracy, please consider the following: operator consistency, pipettes are calibrated “to deliver” and not to “contain”, and pipettes should be calibrated at periodic intervals.
- See more at: http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/cell-culture-videos/the-micropipette.html#sthash.DZ3fhz4x.dpuf

Basics of primer design

Designing Infusion cloning primers

InFusion cloning technology shown in attached file.

First step is to make your construct in silico (may be in Geneious) exactly the way it should look after cloning those two fragments. Then choose forward and revererse primers taking about 15bp upstream and downstream of the two junction regions between gene and vector.

Order primers.

What is PCR?

Setting up a PCR reaction

Making and running an agarose gel

To make a 1% agarose gel:

 

Weigh out 1g of  Agarose 

​

Add to 100ml of autoclaved 1x TAE 

​

Microwave the solution in order to dissolve the agarose (be careful agarose solution!)

​

Let cool to 60C

 

(at this point ~1-2ul of 10mg/mL EBr may be added : soaking the gel in Ethidium Bromide later will not be necessary)

 

Pour into the electrophoresis apparatus

 

After gel has solidified, pour the 1x TAE into the gel box, and remove comb.  Samples are loaded into the gel only after  the buffer solution is in the gel box covering the gel. 
  

Gel Extraction of DNA (MN)

Infusion Ligation Reaction

Procedure:
Infusion reaction:
In general, good cloning efficiency is achieved when using 50-200 ng of vector and inserts respectively, regardless of their length. More is not better. If the size of the PCR fragment is shorter than 0.5 kb, maximum cloning efficiency may be achieved by using less than 50 ng of fragment.

Set up the In-Fusion cloning reaction:
5X In-Fusion HD Enzyme Premix 2 μl

Linearized Vector 3 μl*

Purified PCR Fragment 5 μl*

dH2O (as needed) 0 μl

Total Volume 10 μl

*For reactions with larger volumes of vector and PCR insert (> 7 μl of vector + insert), double the amount of enzyme premix, and add dH20 for a total volume of 20 μl.
Incubate the reaction for 15 min at 50C, then place on ice. Continue to the Transformation Procedure (below): 

pET vector system manual

Transformation of plasmid DNA into competent E. Coli cells

Material and Reagents

  • LB

  • 1.5 mL microfuge tubes

  • 42° C dry-bath

  • Ice

  • 37° C shaker

 

Protocol

 

    1. Thaw competent cells on ice. 20–200µL per tube

    2. Add max.4µL of pure plasmid DNA or of Infusion ligation reaction

    3. Mix very gently (tap on tube walls)!

    4. Incubate the tubes on ice for 30 min

    5. Heat shock the cells for 90 sec at 42°C

    6. Place the tubes immediately on ice for at least 2 min

    7. Add 900µL of LB medium to each tube

    8. Incubate for 1 hour at 37°C and shake vigorously (225 RPM)

    9. Spin down briefly (4100 rpm for 10 minutes) and remove most (~850uL) of the supernatant

    10. Resuspend cell pellet with the rest LB medium in the tube by pipetting up and down

    11. Streak the suspension on a LB agar plate (containing the appropriate antibiotic as the attached picture shows.

    12. After streaking, incubate the plates overnight at 37°C

Plasmid miniprep protocol using Sigma Genelute Kit

Making a long term stock of bacteria

Pick a single colony of the clone off a selective plate and grow an overnight culture in the appropriate selectable liquid medium (3-5ml).

  1. Enter all the relevant information (e.g. vector, strain, date, researcher, etc.) in the online Parashar lab inventory inventory at Quartzy.com. This will also be a good time to record the strain information and record the location. Label strain numbers as VPXXXa and VPXXXb where XXX are laboratory's strain numbers.

  2. Take two cryogenic vials and label them with the XXXa and XXXb internal strain numbers.

  3. Add 333 micro liter of 60% glycerol in H2O to each cryogenic vial.

  4. To each cryogenic vial, add 666 micro liter sample from the culture of bacteria to be stored.

  5. Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.

  6. Put in a box corresponding to sdata entry in inventory  and store it at -80C freezer.

More info at:

http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria

ChangeIT mutagenesis short protocol

ChangeIT mutagenesis long protocol

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