Week 5 - 2015

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) and non-ligated or linear DNA ligation products. At this point it is impossible to screen the products because each ligation reaction involves only one set of molecules. We need to create multiple copies (clones) of each ligation product in order to ‘see’ it.

An easy way to make copies of the products is to use E. coli to replicate them. We get the ligation products into the E. coli cells using transformation. Earlier this semester, we made competent E. coli cells. This week we will transform some of our stored competent cells with the ligation reactions from last week. Following transformation the E. coli cells are grown, and if they took up a replicatable ligation product (i.e. the product must be circular and have an origin of replication) the ligation product will be replicated.

Read before coming to the lab

Protocols

  • General guidelines for molecular biology labs
  • Preparation and transformation of competent bacteria
  • Details on how to set up HindIII and BamHI restriction digests are available from Thermoscientific. You looked up this information for Lab 1, please make sure you bring this information to today's lab. Remember to look up the conventional restriction enzymes, NOT the FastDigest enzymes.

To do today:

  • complete the last step in the ligation reaction (heat inactivate the ligase)
  • transform competent cells with your ligation reactions
    • Use the competent cells you made a few weeks ago and follow the protocol, Preparation and transformation of competent bacteria: calcium chloride method - using frozen competent cells.
    • A positive control is not necessary as we already did this control when we made the plates; however, you should still do a negative control.

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 only plate each dilution once (rather than in triplicate) as we are not calculating TE.

To do tomorrow:

Someone 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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