Initial PCR Screen 11-26-12

2015-01-13

azim58 - Initial PCR Screen 11-26-12

I plan to verify the top clones that bound to tumor antibody more than
naive antibody on the array. I will do the verification using PCR.

Primers used found here
Ordered primers for validation 11-19-12

I may use my library 4T1 cDNA, some other 4T1 cDNA from Shen, and some
cDNA from some other mouse tissue.
I should check beta actin as a control in the PCR.
I think I will use dynazyme as the polymerase.

Tumor cDNA from Shen
Item: 4T1 in vivo tumor total cDNA 11-26-12 at Tumor cDNA expression
library 1-20-11 KW -20 C box

For my tumor library cDNA I could use one of two samples.
Purified control cDNA 2-8-11 at Tumor cDNA expression library 1-20-11 KW
-20 C box
or
4T1 Tumor 2nd strand cDNA 3-8-11 Tumor cDNA expression library 1-20-11 KW
-20 C box

I actually had to generate some new 2nd strand synthesis cDNA.
4T1 Balb/c tumor cDNA purified 11-27-12

I made some 1st strand cDNA from a BALB/c mouse ear
First Strand cDNA BALB/c Mouse Ear 11-27-12 KW at Tumor cDNA expression
library 1-20-11 KW -20 C box

3 Samples screened
First Strand cDNA BALB/c Mouse Ear 11-27-12 KW
4T1 Balb s c tumor cDNA purified 11-27-12 (my tumor cDNA)
4T1 in vivo tumor total cDNA 11-26-12 (Shen's tumor cDNA)

11-30-12
The results showed that my tumor cDNA contained all of the genes screened
at the expected size. However, Shen's tumor cDNA did not. The normal cDNA
also did not contain the genes. Note that the normal cDNA and Shen's cDNA
was made using a different kit. I will try making the cDNA with the
different kits and compare the results.

RNA Samples
6-24-10 Tumor RNA
Skin mouse RNA 7-30-12
4T1 in mouse total RNA 9-24

BALB s c ear skin 1st strand cDNA 11-30-12 KW
4T1 Tumor 1st strand cDNA 11-30-12 KW

4T1 Tumor dscDNA purified 12-1-12
BALB s c skin from ear dscDNA purified 12-1-12

12-12-12
experiments performed on 11-29-12 and 11-30-12 and 12-1-12

Diagram comparing the pcr samples made with the same kit.
C:\kurt\storage\CIM Research Folder\DR\2012\12-12-12\pcr analysis

12-17-12
technical repeat of PCRs performed

experiment performed on 12-17-12
annotated diagram found here
"C:\kurt\storage\CIM Research Folder\DR\2012\12-18-12\pcr analysis\pcr
analysis 12-18-12.svg"

The location of the primers along the transcript can be found in the ape
files in the subfolders here
C:\kurt\storage\CIM Research Folder\DR\2012\11-25-12\cDNA Sequence
Analysis
Note that all of the RNA samples all contain the expected bands for the
fragments amplified with this set of primers. However, the normal tissue
also contains many other bands for the Wfdc17_S primer set which the
tumor samples do not contain. This may be due to the tissue type
difference since the tumor samples are breast tissue and the normal skin
tissue is from the ear of a mouse.

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