Lets get the therminology right

1) you don't clone a plasmid, you construct a plasmid by isolating fragments followed by ligation, or you assemble via PCR. The cloning step is when you transform E.coli and select single colonies which originate from a single plasmid starting to replicate in one bacterium each, thus making hundreds of identical copies (clones) per cell and you have millions of cells in a single colony depending on size.

2) You don't amplify the protein, but you amplify the plasmid when you grow E.coli as colony and later liquid culture, and you test the plasmid by restriction analysis, sometimes PCR, sequencing and probably a combination of these.

3) You will get the desired protein when you transfect mammalian cells with the tested plasmid, and the negative control is the untransfected cell line, and you can do several test transfections with different DNA preps of the same plasmid clone, just to make sure one of the plasmid preps is of high quality and transfects well. The first test is to do a western blot to see if your protein can be detected in the transfected cells, but not the untransfected control. Instead of untransfected cells, you can also transfect with the same expression plasmid without the coding region that you have inserted. Its called empty vector transfection.

But this is super simplistic and usually you need many more controls.... but that depends on your experiment.

Lets get the therminology right

1) you don't clone a plasmid, you construct a plasmid by isolating fragments followed by ligation, or you assemble via PCR. The cloning step is when you transform E.coli and select single colonies which originate from a single plasmid starting to replicate in one bacterium each, thus making hundreds of identical copies (clones) per cell and you have millions of cells in a single colony depending on size.

2) You don't amplify the protein, but you amplify the plasmid when you grow E.coli as colony and later liquid culture, and you test the plasmid by restriction analysis, sometimes PCR, sequencing and probably a combination of these.

3) You will get the desired protein when you transfect mammalian cells with the tested plasmid, and the negative control is the untransfected cell line, and you can do several test transfections with different DNA preps of the same plasmid clone, just to make sure one of the plasmid preps is of high quality and transfects well. The first test is to do a western blot to see if your protein can be detected in the transfected cells, but not the untransfected control. Instead of untransfected cells, you can also transfect with the same expression plasmid without the coding region that you have inserted. Its called empty vector transfection.

But this is super simplistic and usually you need many more controls.... but that depends on your experiment.

As the details of the steps have been explained above, I will just provide information for the controls of the experiment:

1. Construction of Recombinant Plasmid:

a. First off, check whether both of the restriction enzymes are working if you plan to do a traditional directional cloning. [Expected Result: Both the enzymes should show linearization of the vectors]

b. Once confirmed, move on to the double digestion of both vector (Plasmid) and your gene of interest.

c. Perform ligation for vector and interest in the molar ratio of 1:3 or 1:6 at either 4 degree or 16 degree centigrade overnight (although there are several kits that perform 15 minutes rapid ligation, choose as per you feasibility).

http://www.insilico.uni-duesseldorf.de/Lig_Input.html

Note: Make sure to keep one single digested vector as well for ligation to be used as a control for later experiment.

Note: Make sure that your plasmid has both bacterial as well as mammalian origin of replication as well as mammalian promoter otherwise you'll have to clone the promoter as well.

2. Transformation Controls: Plates with

a. Positive Control: Usually a vector that has been routinely used in the laboratory and is known to have a higher transformation efficiency. [Expected Result: colonies should appear]

b. Negative Control: Wild type E. coli kept in the media containing the selection marker, to ensure there has been no contamination during the transformation experiment.

c. Ligation Control: Previously ligated single digested vector so as to ensure that ligation was successful. [Expected Result: Colonies should appear]

d. Double Digested Vector: Previously Double Digested vector that has undergone the ligation procedure so as to ensure that complete double digestion had occurred during restriction. [Expected colonies: No colonies should appear]

e. Plain medium with no selection : To be plated with E. coli that has undergone the full procedure of Transformation to ensure that transformation was not deleterious for the bacterium. [Expected Result: Bacterial Lawn should appear]

f. Experiment : Plates containing bacteria transformed with the ligation mix containing both vector and insert. [Expected Result: Colonies should appear]

Note: The experiment can be carried forward with just the positive and negative control, the other controls are just for troubleshooting later if there is no colony or false positive colonies.

3. Identification of the Transformed colonies:

You can start with a colony PCR but sometimes its simply too confusing as multiple bands appear and some could be closer to your length of the gene of interest.

In such cases you can start with the plasmid isolation. Once isolated you can confirm by:

a. Restriction Digestion

i. Single Restriction Digestion:

Using only one enzyme. [Expected Result: Gives a base shift, when compared with the wild type plasmid. i.e. increase in size of the plasmid]

ii. Double Digestion:

Using two enzymes previously used for cloning. [Expected Result: Insert release i.e. two bands appear corresponding to the size of your vector and insert during gel electrophoresis].

b. PCR:

Done using primers of the gene of interest.[Expected Result: Band of Interest should appear at the corresponding size].

4. Confirmation for the gene of interest:

Needs to be done for ensuring there are no unwanted changes in the gene of interest. Since its a directional cloning, normally the genes have the correct orientation for expression.

5. Transformation into the mammalian cells:

Use any of the methods for transformation and you may use positive and negative controls as mentioned earlier. Make sure to grow wild type cells in normal media as well.

6. Once transformation has been completed, let the cells grow under selection media to ensure that only those cells survive that has been transformed.

7. Confirmation of the expression of the transgene:

a. Isolation of RNA:

Isolate the RNA from the transformed and non-transformed cells. Perform Reverse transcription and conventional PCR using the gene specific primers,which should give you the band of interest.

Note: If you are using a gene that has an endogenous level, in other words, if you are trying to over-express the already present protein, you can move on to the real time PCR to see the over-expression pattern.

Note: Although good, sometimes mRNA expression does not co-relate with the translation, hence, you will have to do the protein expression test as well.

b. Isolation of Protein:

Protein isolation from the transformed as well as non-transformed cells, and perform western blotting to see the over-expression pattern.

c. Immunofluorescence:

Apart from the over-expression, you may perform immunofluorescence to see the localization of the newly synthesized proteins with the cells if it is intracellular protein.

Although there are lots of techniques to confirm your results, these are some of the basic ways to do so.

Hope it helped.

The minimum requirement for any experiment is to have both a positive control (PC) and a negative control (NC). A PC is the setup which you know will produce positive results, and a NC is one which you know will produce a negative result. Once you have those two controls, you can think of and set up additional controls, depending and specific to your experiment. A tricky problem to control is the presence of foreign or non-specific inhibitors in your setup, because when that is so, even your PC will give negative results. To make sure there are no such inhibitors, you should make sure that all your reagents and specimens are pure, clean, and fresh. Just my two cents.

If the protein for expression is from a different species than the cells used for expression I would also consider codon optimization. By selecting codons for common tRNAs in the expressing cells you can greatly increase your levels of expressed protein.