Cloning and expressing a mystery 71KDa protein
Posted 10 June 2011 - 08:43 PM
Here is what his instructions to me are:
MOL WEIGHT of protein: ~71 KDa. Allow for 300bp of additional flanking DNA.
100ng of lyophilized (freeze-dried) unknown sample DNA.
Forward Primer: 21bp, G/C content=48%, 10ÁM in H20
Reverse Primer: 21bp, G/C content=48%, 10ÁM in H20
1. Isolation of a supply of the target gene in at least MICROGRAM
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:
Initial denaturation: 94║C for 30 seconds to 2 minutes (see Notes, page 2)
25ľ35 cycles of:
Denature: 94║C for 30 seconds
Anneal: 55░C for 30 seconds
Extend: 68║C 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?
Posted 12 June 2011 - 09:28 AM
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...
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
Posted 15 June 2011 - 06:56 AM
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
Posted 19 June 2011 - 11:08 AM
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.
Posted 20 June 2011 - 02:31 AM
but if you afraid, try to dilute your template a little...then from the diluted stock try make another dilution...
Posted 20 June 2011 - 04:51 PM
Afterwards, the secreted protein will be purified via batch purification with a HIS-tag and Ni column.
Posted 20 June 2011 - 06:06 PM
Posted 20 June 2011 - 07:22 PM
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
Posted 23 June 2011 - 06:19 PM
Problem is the protein is a transmembrane protein. We'll see how that goes.
Posted 02 July 2011 - 10:17 AM