UNFOLDING THE MYSTERIES OF GENETIC SCIENCES IN INCURABLE DISEASES LIKE CORONARY ARTERY DISEASES, CANCER, & RAREST OF THE RARE DISEASES LIKE MOYAMOYA, SLE, TAKAYASU ETC.

         Move Next  >>

Job Requirement:

        RNA Isolation using GeneiPure Total RNA isolation Kit from Blood (cat No. 117688)

        Amplification of gene 1 endoplasmic reticulum golgi intermediate compartment protein (ERGIC) by RT PCR

        Cloning and bi-directional sequencing

        Analysis of sequence

 Steps Involved:                                        

1.   RNA was isolated from fresh blood as per the instructions manual of the Kit

2.   Standardization of RT-PCR for the Gene 1(ERGIC) amplification (size 818 bp)

 Synthesis of cDNA performed with GeNei M-MuLv RT-PCR kit (cat no.105593) as per the given protocol using oligo    dT primer 

PCR Conditions:

Step 1              94^oC for 2 min

Step 2              94 ^oC for 30 sec

Step 3              54 ^oC for 40 sec

Step 4              72 ^oC for 60 sec

Step 5              32 times repeat step 2 to step 4

Step 6              72 ^oC for 10 min

Step 7              4 ^oC hold

3.  The PCR products were resolved on 1.5% agarose gel (Fig.1) and purified using GeNei Gel Extraction Kit  (KT-93)

4.  Purified PCR products cloned into pGEMT®-Easy vector.

5.  The clones were confirmed by release of insert using EcoRI restriction endonuclease from pGEMT®-Easy (Fig.2)

6.   Three clones given for bi-directional sequencing with M13 forward and reverse primers