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.This week we will transform competent DH5α cells with the ligation reactions from last week. Following transformation the E. coli cells are grown, and if they took up a replicable 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
- Project overview – This is a good time to review the entire project, focus in particular on Step V. Screening the newly constructed plasmids.
- Blue-white screening
Protocols
- General guidelines for molecular biology labs
- Preparation and transformation of competent bacteria
- HindIII and BamHI FastDigest protocols; available from Thermoscientific. You looked up this information previously.
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).
- A positive control to test the transformation efficiency of the competent cells and a negative control.
while the cells are incubating
- go over Predicting possible recombinants and decide how many colonies should be screened for each ligation strategy
- 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 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.