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.

Notes

yet to finish the typing part of the project.

Sravan Kumar.Galla

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