Week 2

Determine quality and concentration of digested plasmids

Before using our digested DNA in the upcoming cloning steps we need to confirm that the digest is complete and we need to know how concentrated the digested DNA is. We will determine both concentration and quality by running a small amount of each digested plasmid on an agarose gel.
Once we know the concentration, we may need to set up new digests if either or both plasmids is not concentrated enough, or not high quality.

Read before coming to the lab

  • Project overview - Step III - Produce and prepare the DNA fragments for cloning.

It is a good idea to review ALL project steps each week before coming to lab. This will help you get a better handle on the whole project as we move along.

  • Working with DNA volumes and amounts - practice problems. We will be doing several calculations that involve working out what volume of a DNA solution will give a specific amount of DNA. This is often difficult for newbies, so make sure to try the practice problems.
  • All protocols listed below.


To do today:

Run agarose gel

  • a prepared 25 ml, 0.9% agarose gel (1X TAE) with 0.1 µg/ml ethidium bromide, will be ready for you when you come to lab

Note: To calculate a precent-weight-per-volume (agarose is a solid and is weighed but TAE is a liquid), calculate the % of the volume in ml and convert to a gram weight (e.g. to make 1% gel in 100 ml you would use 1 g of agarose)

  • prepare 2 µl of each digest for loading on the gel (do not add loading dye directly to the digests) - see Quick guide to preparing and loading samples
    • Note how you will prepare samples for loading (we will check this before you start)
  • set up the gel running apparatus
  • load gel with 5 µl of prepared 1KB DNA [ ] ladder and each groups’ prepared digest samples (place the ladder between the 2 groups samples)
  • run gel for 30 – 50 min at 80 volts

While the gel is running, work out the amounts of digested pKC7 and pUC18 that you are going to need for ligations (see below).

  • photograph gel
  • determine the digested plasmids’ quality (look for complete digestion and the correct sized fragments)

The plasmids must be completely digested and sufficiently concentrated for the steps we will carry out in the next few weeks; therefore, if you have any incomplete digests this week (or any other problems with your digested plasmid DNA) we will need to fix the problem before next week. The lab instructor will go over your results with you today and give you direction on what to do if there are any problems. Fixing the problem may involve coming back outside your scheduled lab time.

  • Determine the digested plasmids fragment sizes and the concentrations of each digested plasmid. Remember to record this in your notebook, and explain how you arrived at each estimated value.

Prepare for gel purification of Gene A: do you have enough digested pKC7 for gel purification?

Work out the total amount of Gene A you need to purify (see protocol book for how to do this).
Keeping in mind you will loose 20 – 30% during the purification do you have enough Gene A in the digest to get purify the amount you need?

amount of Gene A needed for gel purification step

[digested pKC7] ___

[of Gene A in digest] _

amount of digested pKC7 that will give you the amount of Gene A you need to gel purify _

Prepare for ligation reactions: is your vector (pUC18) concentrated enough?

We are using T4 ligase from Thermoscientific. We will be setting up 20 μl ligation reactions and will aim for the midpoint amount of vector (see the manufacturers product information to find the recommended amount of vector needed in each ligation reaction).

Recommended amount of vector needed in each ligation reaction =

You can only add 3 or 4 µl of digested pUC18 to the ligation – will your pUC18 concentration be high enough to get the desired amount of pUC18 in the ligation?

desired amount of pUC18 in each ligation reaction =

[digested pUC18] =

volume of digested pUC18 needed=

Preparation for shotgun ligation: what volume of digested pKC7 is needed?

For the shotgun ligation we will use 1:1 molar ratio of digested pKC7 to digested pUC18

desired amount of digested pKC7 ___ ng

[digested pKC7] =

volume of digested pKC7 needed to get the desired amount _ μl

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