Search found 22 matches

by siddharthsameer
Tue Jul 17, 2012 9:14 am
Forum: Molecular Biology
Topic: restriction analysis
Replies: 14
Views: 15103

Re: restriction analysis

hello Thanks a lot for your reply one silly doubt once i do the PCR with the vector and then I do the gel electrophoresis , so when I do the gel electrophoresis I should get the band of my desired gene (Suppose my desired gene is of 1428bp) and then I cutthe band and purify it.. is that my cocnecot ...
by siddharthsameer
Mon Jul 16, 2012 3:56 pm
Forum: Molecular Biology
Topic: MOLECULAR CLONING
Replies: 9
Views: 11174

thansk a lot
by siddharthsameer
Mon Jul 16, 2012 12:33 pm
Forum: Molecular Biology
Topic: restriction analysis
Replies: 14
Views: 15103

Re: restriction analysis

thanks a lot I understood little bit , but in my case as told to me that the ordered nucleotide sequence will be incoroporated with the plasmid and then i have to to subclone it into E.coli TOF competent cell and then i have to plate it select for the colonies and then do the mini prep to get the pl...
by siddharthsameer
Mon Jul 16, 2012 7:54 am
Forum: Molecular Biology
Topic: restriction analysis
Replies: 14
Views: 15103

Re:

What's your vector name? Do you have sequence or restriction map of it? hello My nucleotide seuence is of 1428bp and my vector is pPICZalpha A and its about 3,3kb . if i do the restriction digestion with ECOR1 and Kpn1 FOR MY insert (gene of my interest) and my Vector, what is the actual observatio...
by siddharthsameer
Sun Jul 15, 2012 8:12 pm
Forum: Molecular Biology
Topic: restriction analysis
Replies: 14
Views: 15103

Re:

You have to find out, where your REs cut. Let's say it will be in positions 350, 1400 and 3050. Thus you will have fragments 1050 (1400-350), 1650 (3050-1400) and 600 (3300-3050+350). thanks for the reply but i didnt get anything, rather i got confused kindly explain me brefly.. sorry to trouble yo...
by siddharthsameer
Sun Jul 15, 2012 11:55 am
Forum: Molecular Biology
Topic: restriction analysis
Replies: 14
Views: 15103

Re:

From the gel you simply read sizes of your fragments and then check whether is it the same as expected from in silico / in papiro :) prediction of your restriction. For that you either past your sequence into some program and selects appropriate REs and it will show you the result or make a calcula...
by siddharthsameer
Sun Jul 15, 2012 11:37 am
Forum: Molecular Biology
Topic: MOLECULAR CLONING
Replies: 9
Views: 11174

Re:

JackBean wrote:usually the insert is in 3-times molar excess

thanks for the reply but i want to undertand the complete calculation to find out the volume for insert and vector.. can u help me in that context?
by siddharthsameer
Sun Jul 15, 2012 10:24 am
Forum: Molecular Biology
Topic: MOLECULAR CLONING
Replies: 9
Views: 11174

MOLECULAR CLONING

HELLO EVERYONE I am about to start my cloning and I would like to know a proper calculation for the ligation (Insert to vector). I saw many calculation but could not understand that , I would like to know how do you calculate the volume needed for the insert and the vector.. I am really troubling wi...
by siddharthsameer
Sun Jul 15, 2012 7:24 am
Forum: Molecular Biology
Topic: pichia pastoris
Replies: 1
Views: 2508

pichia pastoris

hi all
Can anyone tell me about the expression in pichia pastoris..Is it the same like eukayote? that is from the nucleus to cytoplasm ,i am little bit confused if someone has worked on it kindly help me.
regards
sameer
by siddharthsameer
Sun Jul 15, 2012 6:54 am
Forum: Molecular Biology
Topic: restriction analysis
Replies: 14
Views: 15103

restriction analysis

hello all i am a new student of molecular biology and i have few things to ask i know these are silly questions to be asked but i am unable to find the answer. 1.how do i interpret the agrose gel result of the PCR and restriction double digestion on the basis of marker. i really dont know how to do...