Basics of cloning - HELP! - I'm new to this and need some help! (Sep/23/2010 )
I'm new to molecular biology and am trying to grasp the basics of cloning. Basically, I am in a new lab and would appreciate any help because there is no one here to help me.
I need to transfect hec cells with a pcdna plasmid, but first I have to prepare the plasmid. I have done transfections before so that part is ok, but it's the prep that I'm worried about.
I have the sequence of interest in a bacterial plasmid and I need to get it ready for transfection (ie clone it into the pcdna). How do I do that? i know it requires cutting it out, then purifying? maxi prep? ligation?
I'm really lost!
any help very appreciated.
so u hav long way to go!!..fine now forget abt transfection, u have to clone ur insert which is in another vector into Pcdna....to do that u have two ways one is to study the bacterial plasmid, in what way ur insert has been cloned, i mean using wat enzymes they hav cloned ur insert...if u know tat and if the same enzyme is present in pcdna MCS in right orientation u can directly cut the insert gel purify and cut Pcdna also wit the same enzymes gel purify and do ligation... another way is that u have to Pcr ur insert frm baterial plasmid with Restriction enzymes on both primers and ligate into the pcdna as above..then transform ur ligation in good competent cells and pick some around 10 colonies, do mini prep and screen for insert....for screening u hav two ways, either thru restriction digestion or PCR....if u get positive for insert do a maxi using tat mini inoculum and now u r ready to do ur transfection...may be u ll get more ideas in this forum....good luck get back if u have any other queries....
Gnana...
thanks for the reply, very helpful. More details would be great! If i understand correctly:
cut insert - gel purify
cut pcdna - gel purify
then ligate the insert and pcdna
then now i guess i have to check whether it was inserted properly right? I should run a gel and compare?
Then I have the insert hopefully in the plasmid and I have to somehow expand it, right? Grow it on bacteria? (i think people somehow streak it on? i guess i have to put it in lb media?), select colonies, maxi prep, test concentration and then tranfect? (fyi i have to transfect, ie. not transform because of our system)
any info on the protocol of getting the final product would be appreciated!
s u have to check ur insert for orientation if u cloned with single RE. u can check the orientation by digestion for example select an enzyme in the plasmid and look for the same or other single cut enzyme in ur insert and do digestion, now u can asses the size of ur digested product if it is in right orientation or in otherway also..so by this way u can select the right clone...
usually u would do a mini inoculation (5ml)overnignt and from that u ll take 1 ml or 2 and do mini prep and do all the things i said above,,,once u got the right clone u expand that clones remaining 3 ml in 250 ml LB with selection and do a maxi prep, tats it...
gnana..
First of all... what plasmid is your insert currently in? It may well be in a mammalian expression system already.
There are lots of protocols for subcloning in the links on the "protocol online (www.protocol-online.org)" part of this website. It would be a good idea to read some of them. If you can find a book (3 volumes) called "Molecular cloning: a laboratory manual" by Sambrook, Fritsch and Maniatis, it will be a great help. Especially if you can find the most recent (2004?) edition, which has updated protocols using more modern reagents.
Your basic steps are:
1) cut the insert out of the parent plasmid using restriction enzymes that are compatible with the plasmid you are cloning into. This will be best done with two different RE's so that you can clone directionally. Be aware that you need to keep the insert in-frame so that it codes properly Purify the insert by gel extraction.
2)Digest your receptor plasmid with the same REs you used in step 1 - this will give you compatible ends. You should be able to purify the insert by using a PCR clean up kit, or you can gel extract.
3)Ligate the insert and plasmid using T4 DNA ligase. Use 1:1 ratio of the two if you are doing directional cloning.
4)transform ligation into competent cells. Chemically competent cells are the easiest to use with heat shock.
5)Plate the ligation out by spreading on a selective plate with appropriate antibiotic. Incubate at 37 deg C overnight. You should have individual colonies on the plate.
6)Pick some colonies (10-20 should be enough) into 2-5 ml of the liquid version of the same medium as you plated on with the same antibiotic.
7)Grow cultures and perform a miniprep using alkaline lysis. Also make a glycerol stock and store at -70 to -80 deg C
8)sequence the insert region to determine if the insert is in place and correct orientation, correct sequence, etc.
9)Streak a small amount of the correct glycerol stock (don't defrost, just scrape the surface) onto a fresh plate, pick, grow, make a bigger culture and perform a midi or maxi prep which can then be used for transfection into mammalian cells.
As you might have noticed to get from where you are to where you want to be is a long process. You will need to have a postdoc or your supervisor hold your hand while you go through the steps. AS you might have notice from the many post in this forum, thing can go very wrong.
Roughly the things you need to do are
1 - Get your book keeping done.
Get the DNA sequence of all the plasmids that you have. (pCDNA, plasmid with bacterial gene) and place it into sequence manager such as DNAstar or VectorNTI. The sequence manager program will tell you what Restriction sites are present in the plasmid and help you keep tract of where all the parts are. Do not go building a plasmid without having a clear idea of what you have and how to get there. Mistakes in planing are very costly.
2- Verify that the plasmid with the sequence of interest really is what it is suppose to be.
People make mistakes and you might have been the wrong plasmid or the gene of interest could have a mutation.
3- Execute the construction plan.
The general outline has already been discussed by others in this thread.
4- Verify the plasmid is correct and without mistakes
5- Transform.
I was new to all this a month or so as well, don't worry these things do take time! (and in my case several threads on this forum...)
I found this NEB cloning guide very useful (although they link to all their products obviously, most products there can be bought from other companies as well, most of mine was Promega)
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cloning_guide.asp
And I found this free website to be very useful in terms of organising DNA sequences of both plasmids and inserts, and identifying unique restriction sites. It also has a plasmid designer tool which will automatically work out primers and restriction sites for you, In terms of time saved this website is great, and is free!:
https://www.lablife.org/
The advice the others have given here is good too, just don't be afraid to ask for advice.
thanks a lot everyone! I appreciate the help and advice. I will be starting at the end of the week so I"m sure i'll be asking many more questions. Also thanks for the link "Enthusiast".
