Saturday, 16 July 2016

Before Cloning….

For using restriction enzymes to digest and ligate genes of interest into plasmid vectors, it is good practice to check if your restriction enzyme(s) of choice also cuts at regions other than those intended. For standard commercial vectors, these usually have a multiple cloning site (MCS) region containing unique restriction sites where the corresponding enzyme will only cut. However, if you opt to use an enzyme whose site is not present in the MCS, you will need to check if your enzyme(s) of choice cuts anywhere else on the vector.

For your insert, if you decide to engineer restriction sites onto the 5’ and 3’ ends, it is better to (1) choose sites that do not cut anywhere in the insert and (2) make sure the restriction site you are incorporating correspond to what is present in the MCS of the vector.

Some good online tools that are freely available to use for identifying restriction sites in DNA sequences include:


Saturday, 2 July 2016

Calculating the Geometric Mean/Geomean of Housekeeping Genes from Real-Time PCR Data Using Excel

In real-time PCR, it is not uncommon for multiple housekeeping genes to be used for normalising data. Knowing how to calculate an average for your housekeeping genes will be useful regardless of whether you opt to carry out relative or absolute quantification of your gene of interest.

If you are using three or more housekeeping genes, you can calculate the geometric or geomean easily using Excel.





Cell Formulae
Column B: Gene 1 CT values from your qPCR run
Column C: Gene 2 CT values from your qPCR run
Column D: Housekeeping gene 1 CT values from your qPCR run
Column E: Housekeeping gene 2 CT values from your qPCR run
Column F: Housekeeping gene 3 CT values from your qPCR run

Column G: =GEOMEAN(D4:F4) and copy/paste formula down to G18