User:Sravan Kumar Galla/Notebook/Transcriptional Analysis of Claudin 4 Promoters

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Project Description/Abstract
'''Human Claudin 4 Promoter Project:

TRANSCRIPTIONAL ANALYSIS OF PROMOTER 2, 3, 4 AND 5:'''

Promoter constructs Protocol:

1.	PCR: Picked Primers for each of the promoter and performed a Touch Down PCR.

2.	Gel Electrophoresis: Performed to see the PCR product. Used Sybr Safe and not EtBr.

3.	PCR Product Purification: S.V Gel Purification kit used to purify the PCR product.

4.	Taq Therapy: Purified PCR product subject to Taq therapy.(Kit used) PCR Product  40µl 10xBuffer     5µl Mgcl2 25mM    3µl dNTP's        1µl Enzyme        1µl Incubated @ RT for 10 mins - 15 mins

5.	TOPO cloning: (pBLUE TOPO KIT Used) PCR Product    4µl Salt Solution  1µl Vector         1µl Incubated @ RT for 30 mins. Tubes transferred to ice immediately.

6.	Transformation: Kit Protocol Vector(from above step)  2µl Competent E coli cells  25µl Subjected to heat shock for 30 seconds @ 45 C Cells/Tubes transferred to Ice immediately and incubated for 15 mins to 45 mins. Longer incubation time did not show any change of the results. 250µl of SOC added to each tube and incubated on shaker for 1 hr @ RT.

7.	Platting (Made LB Plates with Amp): Plates were pre-heated for 30 mins in the oven. After 1 hr of incubation SOC with cells were platted on LB with 75µl and 250µl each.

8.	Clone Check PCR: a.PCR master mix of 450µl made. Master Mix: Nuclease Free Water  337.5µl 10X PCR Buffer         45.0µl MgCl2 (25mM)           27.0µl Forward Primer (10µM)  13.5µl Reverse Primer (10µM)  13.5µl dNTP's                  9.0µl Enzyme (Taq)            4.5µl Total Vol.            450.0µl Labeled 8 PCR tubes and added 50µl of master mix in each. Grid Plates used for growing the colonies from master plate and second plate. From the Master plate picked a colony with a pipette tip and streaked as single colonies (dotting them diagonally{3 picks}) The same tip placed in the PCR tube and incubated @ RT for 3mins to 5mins. The same was done for the rest of the colonies and the tubes. PCR was performed (Touch Down PCR) Product was confirmed by running the samples over the gel.

9.	Mini Prep’s

10.	Nano Drop

11.	Sequencing

Promoter Construct 2: No Mutations seen in the sequence Glycerol stocks made and stored Plates stored at -20.

Promoter Construct 3: High mutations seen Glycerol Stocks made and stored @ -4 Plates stored at -20

Promoter Construct 4: No mutations seen in sequence Glycerol Stocks made and stored @-4 Plates stored at -20

Promoters construct 5: Promoter could not be cloned and transferred into the vector. Trouble shooting done but still could not be cloned in. As per Dr. Frank moved on to Cell Line Screening.

''' CELL LINE SCREENING:

A549 CELL ASSAY:'''

a.	A549 cells sub-cultured and seeded 60,000 cells.

b.	PMA Treatment (PMA and DMSO stored in -4

c.	RNA Isolation

d.	c-DNA Synthesis. Human claudin 4 Primers used (Primers previously used by Ying for Real time PCR, stored in -80 (Kary’s Lab))

e.	Samples stored in -80 (Box labeled as RNA Isolation at 4, 6 and 8 hr time point)

f.	DNAase treated samples (6hrs) stored in -20.

g.	4 hour and 6 hour time point analysis of c-DNA done.

h.	4 hr samples show high amount of gene expression

i.	Tried to duplicate the results, but only one PMA sample worked. This might be due to the degradation.

MLE – 12 CELLS c-DNA Analysis:

a.	Time points 4 and 6 with Control, PMA and DMSO used to perform c-DNA analysis.

b.	Mouse Claudin 4 Primers Used.

c.	Samples stored in -80.

d.	PCR samples stored in -4.

e.	Bands seen in all the samples except PMA (1) sample.