Cloning and expressing a mystery 71KDa protein - (Jun/10/2011 )

Hi everyone, I'm new to the forum. Let me explain my situation. I work in a lab with an odd supervisor who runs cloning and expression competitions b/w the undergrads in his lab. He gives you primers, a piece of UNKNOWN DNA and a limited supply of certain plasmids, expression vectors, reagents, etc. to identify your insert, clone it up, determine the correct expression vector, and express your protein and purify it. Afterwards, he performs enzymatic assays to determine if the protein is legit. It is a lot of fun to do this on the side outside of my normal project and very useful since I learn a lot but stressful because he takes everything quite seriously and is quick to express his discontent. So I need help from you guys--advice, comments, tips. I can also keep you guys informed on my progress in these games so you can act as my sideline fans and coaching staff.

Here is what his instructions to me are:


MOL WEIGHT of protein: ~71 KDa. Allow for 300bp of additional flanking DNA.

I HAVE:

100ng of lyophilized (freeze-dried) unknown sample DNA.


Forward Primer: 21bp, G/C content=48%, 10µM in H20


Reverse Primer: 21bp, G/C content=48%, 10µM in H20

MILESTONES

1. Isolation of a supply of the target gene in at least MICROGRAM
amounts.


QUESTIONS:

So to reach my first milestone, I need to PCR my sample product. He only provides PlatinumTaq High Fidelity Taq so I need to use the corresponding protocol.

Here is what I setup thus far-50μl PCR reaction

Component Volume Final Concentration
10X High Fidelity PCR Buffer 5 μl 1X
10 mM dNTP mixture 1 μl 0.2 mM each
50 mM MgSO4 2 μl 2 mM
Primer mix (10 μM each) 1 μl 0.2 μM each
Template DNA ≥1 μl (as required)
Platinum® Taq High Fidelity 0.2 μl 1.0 unit*
ddh20 to 50 μl Not applicable

I want to know how much DNA to add to the reaction. Since I have 100ng total DNA freeze dried, what volume should I resuspend it in? 20μl? 25μl? I want enough leftover so I can run multiple PCRs, if need be.

My insert is 1,917bp (71kDA protein) +300bp (he said allow for 300bp extra)=2217bp.


Also for my PCR rxn,

here is my cycling setup:

35 cycles
Initial denaturation: 94ºC for 30 seconds to 2 minutes (see Notes, page 2)
25–35 cycles of:
Denature: 94ºC for 30 seconds
Anneal: 55°C for 30 seconds
Extend: 68ºC for 2:20second (1 minute per kb of PCR product)

So what should the initial denaturation length be? Let's say my product is 2.3kb, is 1minute,30 seconds fine?
So I want to get AT LEAST 1 microgram of DNA from this reaction. Is this protocol feasible? What would you do?

This is fun.

I not sure how others think, but this is my opinion.

He give you 100ng DNA freeze dry. Try resuspend in 1ml dH2O, that will be 100pg/ul, more than enough template...

I think you should scale down your total volume to 25ul or 15ul... if can only 5ul just to do your gradient PCR.



BTW, he give you "PlatinumTaq High Fidelity Taq"... your boss must be damn rich fellow.... PM me your Boss name & lab... I thought of join his lab...

Hi,

Well, i have a little cheat for you should you wish to take it....

Use the PCR primers to sequence the template, blast it and find your gene that way.

It would be making the most of modern technology and is pretty much what reverse cloning has given way to in the last 5 years or so.

Best of luck

You can't use the pcr primers to sequence the template. I only have 100ng of template. So I would have to use all of my template DNA to sequence it (sequencing service requires 100ng template) for inserts ~2kb. So I need to run PCR to get more template. However, after PCR you have a hypothetical heterogeneous mix of template-because some have mutations. So you need to isolate pure genetic copies--plating and mini/midi preps and then send for sequencing.

I have run the midiprep and have sent the thing for sequencing. I should have the results in on Monday. I have to express and purify the protein from Pichia so I have to choose the correct pPicZa vector and do a double digest-ligation-transformation into e.coli to get a lot of the vector+insert. Afterwards, I will transform it into Pichia and run batch affinity purification with HIS-tags.

I will keep you guys up to date. This should be interesting.

I agree with Adrian K...Use gradient pcr...and you should rmemeber when your template is low, you need to increase the cycle (max is 35) if you have concentrated template 30cycle is more than enough...btw this is interesting race...this is Olympic for molecular biologist hahaha joking!.......and dont forget to do final extension (5-7min)..10, 15 and 20ul template just enough, i have such case where my friend has very little template (genomic extraction) even he got no band if run gel, but when he do pcr the band appear very good... luck for him and lesson for us...

but if you afraid, try to dilute your template a little...then from the diluted stock try make another dilution...

Good luck!

I'm way past the pcr stage. I sent the thing out for sequencing and got my results back. I have many micrograms of the insert in a PCR4-TOPO vector. I found the identity of the insert and will now put the insert into a pPICZalphaA vector and express it in Pichia pastoris once I have gotten microgram quantities of the insert in the expression vector in the right orientation and reading frame.

Afterwards, the secreted protein will be purified via batch purification with a HIS-tag and Ni column.

Hey, how exactly do I check if the insert is in the correct reading frame w/o just sequencing? Is it even possible?

Yeaah you got it:)!!!!

The best one still sequencing...and if you mean to get the orientation on vector, i guess if you using different restriction enzymes, try to cut it and check the fragment...if the size is similar as u expected then it could be your good insert...other way try to pcr and your plasmid(with insert) as a template...if you get desired band it could be good one mean that ok...normally it goes right, if your insert can produce something that can screen in plate is better...by the way sometimes if you pcr especially from genomic template the same size of the band could be appeared as correct but when u go sequencing...it different, so the best one still sequencing...but less worry if u use plasmid

Ok did a digest to confirm orientation of the insert in the vector. Everything is okay. Have midiprepped the expression vec+insert and will transform into Pichia pastoris. Will run batch affinity purification with HIS-tags of the secreted protein.

Problem is the protein is a transmembrane protein. We'll see how that goes.

Ok. The gene is GalNac-T1 and we have managed to affinity purify milligram amounts of the stuff from Pichia. Coomassie stain indicated that the elutions were very clean. Now I will be running an enzymatic assay via the glycosylation of Muc1, one of its substrates.