Progress No 3

It has been a long holiday...and it is not easy to recover.

Anyway, as advised by my supervisor I'll do optimization of the transformation method first before proceeding the yeast transformation with pKLACNA (pKLAC2 + neuraminidase gene). pKLAC1-malE (pKLAC1 + Escherichia coli maltose binding protein) plasmid will be used as the control plasmid for this procedure. Transformation of E. coli with this plasmid was successfully done. So all I have to do now is to choose which method to be optimized and perform the experiment... 

Progress No 2

Yeast transformation - conventional heat shock method, Ito et al 

Since i couldn't get any transformant from the previous transformation by electroporation i decided to concentrate on heat shock method. This method was designed for Saccharomyces cerevisiae, therefore the probability for me to get the tansformant might as well as low as the electroporation. So, after doing the procedure for three times i've finally got a single distinct colony with size ~ 2mm for each pKLACNA and pKLAC1-malE (+ve control: encode for maltose binding protein) transformed cell. In order to confirm whether the colony is a positive transformant or not, i'll have to extract the DNA and do the PCR.

Wish me luck!!!

Progress No 1

x

x

x

Previously:

The H5N1 virus neuraminidase gene (N1 NA gene) has been successfully amplified and cloned into a shuttle vector known as pKLAC2. DNA sequencing confirmed the presence of the NA gene.

Then:

Yeast transformations were done as followed;

1. Transformation  by kit – 3 times
2. Electroporation;
  - Sanchez et al, 1993 – 4 times
  - P. L. Bolen & E. J. McCutchan, 1992 – 4 times
3. Heat shock (Ito et al, 1982)- once

Now:

Still, no positive transformant obtained...