Trouble-shooting PCR with Origene vectors - I cannot get PCR to work, and I have tried many approaches (Oct/30/2007 )

Okay. SO I took over a project involving a vector from origene with a cDNA insert. After troubleshooting the innoculation, growth and plasmid purification/prep, I was ready to tackle the PCR and sequencing. I know the person before me had problems with the PCR, but I thought I would eba ble to manage the obstacles. The first round, I used typical programming, and the enclosed primners for the vector. To no surprise, it did not work. I then tried a PCR with the same programming with my designed primers. All I got were primer dimers. I have been using both my purification product and original vector for comparisons. I tried a touchdown PCR and titrated concentrations of Mg2+, DMSO, and Formamide. Now, my product is too big and won't migrate out of the wells. What should I do now? I appreciate any help, advice, or sympathy.

How about you tell you us what you want to do?

Is this PCR to amplfy cDNA and thus fish out a gene of interest. Or is this a standard PCR, to amplify a gene from a plasmid. Do you know the GC content of your gene, does it have nucleotide repeats in its structure? What are the melting temperature of your primers? What polymerase are you using and what is the size of your desired PCR product. Can you list a typical PCR reaction (conditions and components) that you are using.

Too big? As in you've got too much DNA? Well repeat the PCR and dilute it before you run it out, simple. If you can't get the product, screen the vector by digestion to make sure it is there. If your target has a high GC% (you seem like you are using high GC reagents there) try betaine in your PCR (0.5 - 2M final concentration) - betaine is excellent.

Seeya, Rob

I am trying to pull the cDNA out of a plasmid. My primer Tm is about 60C for all of them. I tried a combination of forward and reverse primers with internal primers, and nothing worked. I used the Expanded High Fidelity polymerase and buffer system. I even titrated levels of MG2. My basic PCR program is...
Step Temperature ˚C Time
Initialization 94-96 2-5 minutes
Denaturation 94 1 minute
Annealing 50 2 minute
Extension 72 2 minute
Final Extension 72 5-10 minutes
Cycled 30 times

And my touchdown...
Step Temp. ˚C Time Cycled?
1 95 2 minutes no
2 95 1 minute yes
3 73 1 minute yes
4 68 2 minutes yes
5 Return to step 2 1 additional cycle
6 95 1 minute yes
7 70 1 minute yes
8 68 2 minutes yes
9 Return to step 6 1 additional cycle
10 95 1 minute yes
11 68 1 minute yes
12 68 2 minutes yes
13 Return to step 10 1 additional cycle
14 95 1 minute yes
15 65 1 minute yes
16 68 2 minutes yes
17 Return to step 14 1 additional cycle
18 68 10 minutes no

I went from primer dimers to a product that stayed in wells. I retried the PCR from the touch down with no avail.
I did do a Not1 digest. The 1st attempt I did on the vectors straight out of the tube. No real results. I redid the digest with products from my bacterial growth and plasmid prep/Purification, and got expected results.

I have not verified the sequence yet. i want to try a PCR on my digest products. I am trying to be optimistic, but I really do not think it will work.
I would love to know how to make this work so I can keep my job (okay so I won't lose it over this), and I wouldl ike to make a great accomplishment since I am new here and the bottom of the totem pole (level and seniority).
I would love any advice.
Thank YOu

Forgive my question, but how do you know your insert is present? You could have empty vector.

So the gene of interest is already in a plasmid?

How big is this product?

Linearising the plasmid/template often helps the PCR reaction.

As this is a sequencing reaction, there is no need for the every or even the majority of the PCR products to be "entirely" correct.
Thus I feel there is no need to use expend high fidelity polymerase (Is this from Roche?) The sequencing gives the average sequence of the population (the bane of polymorphism people). So as long as the average is correct, the sequence will be correct. Even if every molecule has one mutation.

Basic Taq will do okay for upto 900bp. (Which is about the average read length of the average person.) Additionally Taq has higher product yeilds for small product.

I would also:
increase the annealing temperature to 55 Celsius. 10C lower then the melting temperature of the primers feels too much.
decrease he denaturation time to 30 sec (less if the product is 500bp and below)
decrease the annealing time to 30 sec (less if the product is small)
final extention time to 5 mins.
The extention time would probably be lowered too. (~1min)

Lastly, ermm... why can't you sequence directly from the plasmid? It is more straight forward and you don't have to worry about cleaning up the PCR product or making sure that only one band is present for the sequencing reaction.

The products (I have 2 different variants) are 2kbp and 1.6kbp. The main reasons for my PCR is to find primers that work so I can use those for sequencing, and to hopefully find an easy way to amplify my gene, so I have lots and lots. I can send off my eluate from the plasmid prep, but what primers to use? The vector primers supplied by the vector company do not work. The technical support people even told me they do not work. There is also a history of the vector maps not being entirely correct, which makes the sequencing more difficult. Since they are so big, sequencing the whole plasmid also costs much more.
I did a not1 digest, and the inserts are there. I have developed a culture growth protocol to greatly increase my plasmid yield. I just can not figure out how to easily amplify the product or to find primers that work and can be used for sequencing.
I am running a new PCR with new primers with your advised programming modifications. Cross your fingers and toes.

So the gene of interest is already in a plasmid?

How big is this product?

Linearising the plasmid/template often helps the PCR reaction.

As this is a sequencing reaction, there is no need for the every or even the majority of the PCR products to be "entirely" correct.
Thus I feel there is no need to use expend high fidelity polymerase (Is this from Roche?) The sequencing gives the average sequence of the population (the bane of polymorphism people). So as long as the average is correct, the sequence will be correct. Even if every molecule has one mutation.

Basic Taq will do okay for upto 900bp. (Which is about the average read length of the average person.) Additionally Taq has higher product yeilds for small product.

I would also:
increase the annealing temperature to 55 Celsius. 10C lower then the melting temperature of the primers feels too much.
decrease he denaturation time to 30 sec (less if the product is 500bp and below)
decrease the annealing time to 30 sec (less if the product is small)
final extention time to 5 mins.
The extention time would probably be lowered too. (~1min)

Lastly, ermm... why can't you sequence directly from the plasmid? It is more straight forward and you don't have to worry about cleaning up the PCR product or making sure that only one band is present for the sequencing reaction.