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.