Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Cloning strat...Awful or ok?


  • Please log in to reply
10 replies to this topic

#1 uncutlateralus

uncutlateralus

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 28 July 2009 - 06:12 AM

Hello I currently have a cloning strategy in place that I use regulaly. I know it works but its incredibly difficult and it can take months and a huge amount of resources to get even one clone. I'm a phd student in a underfunded lab and i'm given very little freedom protocal wise so would just love some input from everyone.

I clone all my genes into Pet28a expression vector and transform into TOP10 frozen chem comp cells, the procedure is as followed:

1. Amplify genes with KOD polymerase (note that the R.E sites I add are always NdeI and XhoI and i have no overhangs (much to my disgust :P )
2. Clone into PcrBlunt (god knows why we bother with this step)
3. Screen and select positive blunt clone.
4. Prepare blunt clone but 1st creating spin dna (we use a 'cost-effective' alternative to quigen) of uncut clone.
5. Cut with NdeI takes about 4 hours and run on large gel for four hours (depending on band size).
6. Gel extract (also using a cost effective kit, note we are not allowed to use too much columns to even tho it says max 400mg i always work with about 500mg or more).
7. Then cut with XhoI and gel extract(same as before)
8. finally ethanol precipitation to reduce concentration down to about 10ul (usually get very crappy concentrations of around 50ng/ul)

Concerning the pet28a vector it is done exactly the same way using the spin kit (i wish we had a vector kit) if i'm unlucky and not allowed to make my own uncut pet28a I get given about 10ug and from that i can sometimes only get about a total of 500ng once we've wasted hugh amounts during the r.e digestion and gel extraction.

Neways any input into how awful this is would be recommended I'm well aware that this way of during things is awful but i dont really have the input to change it bar slight modifications i can get away with.

cheers

#2 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
16
Good

Posted 28 July 2009 - 05:01 PM

Sound pretty wasteful for a cost conscious lab...

I would do the PCR, double digest the PCR product and the pET28a vector, gel purify both, and ligate. Cuts out all the spin columns, the use of PcrBlunt, half the gel purification work, all the ethanol precipitation, half the clone screening work, etc., plus it turns what is likely a multi-day protocol into PCR in the morning, transformants the next day.

The most expensive thing in the lab is the personnel, squandering both time and reagents without justification is very wasteful and expensive.

#3 social

social

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 28 July 2009 - 09:41 PM

It's so nice to be here. We can lots of things.


taux emprunt
immobilier
- Taux emprunt immobilier.
Comparez les offres d’emprunt immobilier, simulation emprunt
immobilier, taux emprunt immobilier

Edited by social, 29 July 2009 - 12:19 AM.


#4 uncutlateralus

uncutlateralus

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 29 July 2009 - 02:03 AM

Yeah I think it would be much easier to just direct clone into pet vector, the problem i have is that my primers were designed without overhangs so I dont think it would work that way I think you need atleast 6 bp's if you add a NdeI site (i cant afford to order any more).

The thing that bothers me most is using spin dna to make vector as oppose to a quigen vector kit, the vector cuts so poorly that a ethanol precipitation is required to get enough concentration to ligate and even then it only just works (i can get maybe a TOTAL amount of 500ng from 10ug)

#5 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
16
Good

Posted 29 July 2009 - 05:53 AM

Re-ordering the two primers with restriction sites and an appropriate number of 5' extra bases added would cost around $10.00 US total. You would more than make up for this expense the first time you used them to direct clone into the pET vector by eliminating all those other steps (and the costs associated with them).

#6 eldon

eldon

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 63 posts
0
Neutral

Posted 29 July 2009 - 08:16 AM

Re-ordering the two primers with restriction sites and an appropriate number of 5' extra bases added would cost around $10.00 US total. You would more than make up for this expense the first time you used them to direct clone into the pET vector by eliminating all those other steps (and the costs associated with them).


ditto

#7 uncutlateralus

uncutlateralus

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 30 July 2009 - 01:56 AM

Cheers for the advice, I'm gonna try to convince my sups to order new primers....i dont hold out much hope tho!

I've just done some more cloning today and as per usual it was awful and didnt work (i've cloned things before so I know i'm doing it right)

I think the main problem is using spin dna vector as oppose to using dna made from a quigen vector kit, it doesnt cut nearly aswell. Do you guys agree that this is significant issue?

#8 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
16
Good

Posted 30 July 2009 - 03:06 AM

I think the main problem is using spin dna vector as oppose to using dna made from a quigen vector kit, it doesnt cut nearly aswell. Do you guys agree that this is significant issue?


I'm not sure what "spin dna vector" is, so it's tough to comment. But, assuming it's some type of spin column DNA recovery method broadly comparable to the Qiagen spin column kit, its greatest impact should be on the amount of DNA recovered, not on the ability of that DNA to be digested.

The ability of the recovered DNA to be digested would more likely be impacted by what you're using to elute the DNA from the column, assuming the column is stable and doesn't add anything to the eluent.

If you can give us more details on what exactly you're doing at this step, we can probably judge its potential impact better.

#9 uncutlateralus

uncutlateralus

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 30 July 2009 - 04:22 AM

Yeah of course, i'm greatful for sharing your opinion.

Ok so in the past we have used QIAGEN Plasmid Maxi Kit (10) for making the vector and QIAprep Spin Miniprep Kit (50) for making the inserts. Due to cost constrains we now use QIAprep Spin Miniprep Kit to make both vector and insert (although we dont use quigen and use a much cheaper one). we use about 150ml of culture spilt across 15 columns for this and pool the dna at the end. I find using this dna the vector cuts alot less efficently.

In terms of my recovery strategy, I do all additional steps and elute in tris pH 8.0 (i do not add edta). The ellution is run twice (usually i add about 54ul to recover 50ul after two consequative elutions), the elution solution is warmed to 55 degrees and i give 2 mins incubation time for each elution.

cheers.

#10 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
16
Good

Posted 30 July 2009 - 05:35 AM

Okay, there seems to be a lot of false economy assumptions going on here. Let's look at some numbers:


  • QIAGEN Plasmid Midi Kit (100) 100 QIAGEN-tip 100, Reagents, Buffers 12145 $850.00
    $850.00 per kit/100 preps = $8.50 per prep x 1 prep per experiment = $8.50 per experiment.

  • QIAGEN Plasmid Midi Kit (25) 25 QIAGEN-tip 100, Reagents, Buffers 12143 $231.00
    $231.00 per kit/25 preps = $9.40 per prep x 1 prep per experiment = $9.40 per experiment.

  • QIAGEN Plasmid Maxi Kit (100) 100 QIAGEN-tip 500, Reagents, Buffers 12165 $1,742.00
    $1,742.00 per kit/100 preps = $17.42 per prep x 1 prep per experiment = $17.42 per experiment.

  • QIAGEN Plasmid Maxi Kit (25) 25 QIAGEN-tip 500, Reagents, Buffers 12163 $471.00
    $471.00 per kit/25 preps = $18.84 per prep x 1 prep per experiment = $18.84 per experiment.

  • QIAGEN Plasmid Maxi Kit (10) 10 QIAGEN-tip 500, Reagents, Buffers 12162 $199.00
    $199.00 per kit/10 preps = $19.90 per prep x 1 prep per experiment = $19.90 per experiment.

  • QIAprep Spin Miniprep Kit (250) 250 QIAprep Spin Columns, Reagents, Buffers, Tubes 27106 $344.00
    $344.00 per kit/250 preps = $1.38 per prep x 15 preps per experiment = $20.70 per experiment.

  • QIAprep Spin Miniprep Kit (50) 50 QIAprep Spin Columns, Reagents, Buffers, Tubes 27104 $81.00
    $81.00 per kit/50 preps = $1.62 per prep x 15 preps per experiment = $24.30 per experiment.

These are retail prices in US dollars. Your institution may get a discount from these retail prices, so you need to do these calculations with your actual cost plugged in. Include shipping if Qiagen charges you shipping.

However, a general observation is quite clear -- you're using the most expensive method. How does the cost of your "supplier A" kit match up to Qiagen's costs? Moreover, if you're having frequent failures, and one assumes even 50% of these failures would not have occurred if you used the Qiagen kit, the cost per experiment goes up, of course.

Someone really needs to figure out your average cost per experiment (including all steps and reagents used, personnel costs (i.e. time spent on the protocol), and factoring in the cost of the failure rate) of your current protocol versus a more streamlined direct cloning approach. I have a feeling you're currently wasting a lot of money...

#11 perneseblue

perneseblue

    Unlimited ligation works!

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 585 posts
20
Excellent

Posted 30 July 2009 - 08:29 AM

On the point of plasmid extraction from cell culture; With a little practice, I believe the standard alkaline lysis method is only slightly slower that the column binding method. The standard alkaline lysis method is superior in the amount of plasmid DNA you can extract, and it is an fair bit cheaper.

On the subject of columns, it is possible to reuse / regenerate old DNA extraction columns. This would save some money.
May your PCR products be long, your protocols short and your boss on holiday




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.