Week 5 - 2017

Transform competent E. coli with the ligation products

Following ligation, you have a tube of DNA that is a mixture of circular DNA ligation products (possibly more than one type), non-ligated DNA fragments and linear DNA ligation products. We will use a combination of methods to identify our desired recombinant but first, we need to create multiple copies (clones) of each ligation product.

An easy way to make copies of the products is to use E. coli to replicate them. We will get the ligation products into the E. coli strain DH5α using chemically mediated transformation. Following transformation, the cells are grown on solid media and those cells that took up replicable ligation product (i.e. the product must be circular and have an origin of replication) will replicate the products.

E. coli cells can not take up naked DNA unless they are made competent. The cells you will use have been made competent by chemical treatments. However, even following these treatments, only a small proportion of the cells will actually take up DNA. To prevent non-transformed E. coli cells from growing we will include an antibiotic in the plates the transformed cells will grow on. This way, the only cells that can grow are those that took up recombinant molecules that can be replicated (must be circular and contain an origin of replication) and that contain the resistance gene for the antibiotic included in the plate. You will be including a control plate that has no antibiotic. By comparing the growth on this plate to the growth on the plate with the antibiotic, you will be able to estimate approximately what proportion of the cells were transformed.

In addition to the antibiotic selection, the pUC18 plasmid allows us to employ an additional selection technique called blue-white screening. With this screening method, we can easily determine which E. coli took up a regenerated pUC18 plasmid. This step is important for us because we did not purify the large pUC18 vector fragment away from the small pUC18 DNA fragment, thus, in both the shotgun and targeted ligations some of our recombinants will be regenerated pUC18. Make sure you take some time to understand blue-white screening.

Read before coming to the lab

Protocols

To do today:

  • transform competent cells with your ligation reactions
    • Follow the protocol, Preparation and transformation of competent bacteria: calcium chloride method - using frozen competent cells (Lauri has already made and frozen the competent cells but it is a good idea to read through the whole protocol).
    • Calculate the volume of each ligation to add to the competent cells (use the max amount allowed).
    • Use your undigested pUC18 as a positive control to test the transformation efficiency of the competent cells and a negative control.
      • Calculate the volume you will add, again use the maximum amt allowed.
    • When you have writen in your notebook what you are going to add to the cells, we will give you the frozen cells.

while the cells are incubating

  • plan the digests you will carry out on the recombinants (see Making a master mix) – pre-planning the digests will speed up next week's work
  • prepare SOB amp100 plates by adding X-gal as described in “Preparation and transformation of competent bacteria”.

when the incubation is done

  • plate the transformed cells
    • Follow the same procedure used for plating the positive control except for plate each dilution once (rather than in triplicate).

To do tomorrow:

At least one person from each group will need to come in the day after the lab to:

  • remove plates from the 37 °C incubator,
  • check them for colony growth,
  • bag the plates, and
  • store them at 4 °C.

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