2) Combine respective sequences to form contigs.

3) Compare contigs with the accession no. and switch the CDS feature on.

4) Identify fake or true mutations by comparing the graphic overview and the actual hit results.

*Updates*
In the appendices, you will need:
1) standard carves of P1B and P3
2) Hit results of individual sequence result to signify that it is the Mm LDHA gene
3) both F & R contigs with the features annotated
4) hit results (with the CDS features) of the F & R contigs vs accession no; identify where the mutations (if any) fall

They will constitute 11 pages altogether.

In the results, you will need:

1)  the overview graphic snapshot of the F & R contigs vs accession no.
2) a tabulated list of all of the mutations (be it fake or real mutations)

1)      His SpinTrap (GE Healthcare, Singapore)

2)      Bio-Rad Protein Assay (Biorad, Singapore)

3)      InstantBlue Protein Stain (Expedeon, Singapore)

4)      Trans-Blot Turbo Transfer System (Biorad, California, USA)

5)      Anti-6X His tag antibody (Abcam, Cambridgeshire, England)

6)      Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (ThemoFisher Scientific, Waltham, USA)

 

Please bear in mind that all of these questions are meant to be supplementary to your report. I.e., if you already feel that you have sufficient points (after going through the checklist in the guidelines), then it is not necessary to use these pointers below:

 

·         Is there any difference between the non-induced and induced cells? 

o   Purification table parameters

o   Identification of possible LDH band from its size (compare with theoretical MW) on both gel and membrane. 

o   Obvious thickening of the LDH band (on gel and membrane) after induction. 

·         Is there any relationship between the parameters (of the purification table) and the samples? 

·         What is the molecular weight of LDHA subunit? Do you observe a band of similar MW in the other fractions as to the positive controls? How about the elute fraction?

·         How is the purity of the elute fraction (single major band or presence of other bands)? If there are other bands, what can you say about the current purification technique? Is there any way to improve the system (if you are intending to do this, please give known scientific examples to substantiate your claims)?

o   Hint: Purification factor and yield.

·         Do you observe a correlation between the intensity of the LDH band and the specific enzyme activity/purification factor/yield? How do you compare it with the protein assay data?

·         What’s the reason for observing a single band in western blot vs. multiple bands in SDS-PAGE?

·         Is there a difference in intensity of the LDH bands in western blot as compared to SDS-PAGE, especially in the crude extracts? If so, is there any reason to the difference?

·         Do you observe bands in both of your positive controls? Why yes or no?

·         Is the correlation of the band intensities/thickness similar to the previous observed parameters?

·         Do you encounter any “problems” during your experimentation?

o   With protein quantification, enzyme assay data, etc. 

o   With SDS-PAGE total protein staining. 

o   With western blot data (non-specific bands, etc.)

 

Hope this will help you. Good luck in your report writing.

Tomorrow's consultation will be from 10am - 3pm instead because I have something urgent to attend to. I'm sorry. The venue is still the same; ls lab 8.

See you tomorrow.