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Help: How to isolate plasmid from agrobaterium for restriction enzyme digestion - (Dec/16/2007 )

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Hi, everyone. I have construct a plant pexpression vector and plan to transfer plant by agrobaterium. The vector is first transfered into the agrobacterium. Now I have to isolate it from the bacterium and verified it. Usually, I use PCR. But in order to increase the correctness, restriction enzyme digestion was used. However, the concentration of plasmid isolation from agrobacterium by the common methods is not enough to be digestion by enzyme. So does anyone know how to isolate plasmid from agrobaterium for restriction enzyme digestion?
Any assistant is very much appreciated! Thanks!

-tiny1979-

hi

I had the same problem , but what i did was used the same kit or protocol for plasmid preparation and used atleast 20 microliter of plasmid dna for restriction digestion so the bands will be clearer
of course u will have to increase the reaction volume and enzyme buffer volume accordingly but can keep the restriction enzyme concentration same
for example
for a double digestion -with two enzymes having common buffer

Plasmid DNA -20-( can be increased if possible)
10X enzyme buffer-3 ( final concentration 1X , according to the final reaction volume)
Enzyme 1- 0.5U hence 0.5microliter
Enzyme 2-0.5 U
DW- enough to make upto 30 microliter

Overnight digestion
next day run on agarose gel -- very thick gel with the well volume that can hold 30 microliter

hope this works for you
good luck

laxmi





QUOTE (tiny1979 @ Dec 17 2007, 11:05 AM)
Hi, everyone. I have construct a plant pexpression vector and plan to transfer plant by agrobaterium. The vector is first transfered into the agrobacterium. Now I have to isolate it from the bacterium and verified it. Usually, I use PCR. But in order to increase the correctness, restriction enzyme digestion was used. However, the concentration of plasmid isolation from agrobacterium by the common methods is not enough to be digestion by enzyme. So does anyone know how to isolate plasmid from agrobaterium for restriction enzyme digestion?
Any assistant is very much appreciated! Thanks!

-phytoviridae-

If you gel can't hold a larger volume: start from a bigger culture, do 5x (or even more) the miniprep procedure from this sample, pool the extracted plasmid, precipitate to increase concentration and do your digest.

Good luck.

-vairus-

What we usually do is a regular miniprep, then take a couple micoliters from that and retransform it into regular e. coli. Takes a few days longer, but its more reliable.

-smu2-

Thank you for all your helpful suggestion.
Now we can find this is a common problem which we are puzzled.
【What we usually do is a regular miniprep, then take a couple micoliters from that and retransform it into regular e. coli. Takes a few days longer, but its more reliable. 】 We also use this method.
But I wondering if there is a better and efficient method to isolate plasmid directly from agrobacterium and the concentration/quality of the plasmid is available to digestion?

-tiny1979-

Didn't this plasmid start out as an E. coli plasmid? Don't you already have a plasmid prep in the freezer?

You could also consider colony pcr, which would provide a pcr product that could be quickly analyzed by restriction digest, with the added benefit of amplifying only the region you cared about.

-phage434-

QUOTE (tiny1979 @ Dec 19 2007, 02:06 AM)
Thank you for all your helpful suggestion.
Now we can find this is a common problem which we are puzzled.
?What we usually do is a regular miniprep, then take a couple micoliters from that and retransform it into regular e. coli. Takes a few days longer, but its more reliable. ? We also use this method.
But I wondering if there is a better and efficient method to isolate plasmid directly from agrobacterium and the concentration/quality of the plasmid is available to digestion?



I have a protocol for agrobacterium miniprep if you want it. I could never get it to work reliably, but someone else in my lab did so I think its kind of touchy. If you want the protocol let me know.

-smu2-

QUOTE (phage434 @ Dec 19 2007, 04:58 AM)
You could also consider colony pcr, which would provide a pcr product that could be quickly analyzed by restriction digest, with the added benefit of amplifying only the region you cared about.


The plasmid containing goal gene will be transfered into plant. PCR may lead to false positive, which occurred in my t-vector clone. How do you think? And some report even showed that the purity of plasmid would affect the transformation efficiency of plant. Are there anybody give some suggestion about the relation of plasmid and transformation efficiency?

-tiny1979-

I have a protocol for agrobacterium miniprep if you want it. I could never get it to work reliably, but someone else in my lab did so I think its kind of touchy. If you want the protocol let me know.
[/quote]

I am so appriciated for your give of the protocol. Let me try it. And I will tell you the result. Hope the protocol can give us help. Thank you very much.

-tiny1979-

Here's the protocol. Hope it works for you.

1. Grow agrobacterium cells overnight in 1 ml medium containing appropriate antibiotics with vigorous shaking at 28C.
2. Transfer culture to 1.5 ml tube. Centrifuge cells for 1 minute.
3. Discard supernatant, resuspend in 0.1 ml of solution I.
4. Add 50 ul of solution I containing 15 mg/ml lysozyme. Vortex gently for a few seconds.
5. Incubate at room temp 10 minutes.
6. Add 0.3 ml freshly prepared solution II, shake to mix
7. Incubate at room temp for 10 minutes
8. Add 45 ul of phenol equilibrated with two volumes of solution II. vortex gently for a few seconds. Should get very viscous.
9. Add 225 ul of 3M sodium acetate, ph5.2. Shake tube briefly.
10. Incubate at -20C for 15 minutes.
11. Centrifuge for 3 min at 14,000 rpm. Quick pour the supernatent solution into a new 1.5 ml tube.
12. Fill the tube with isopropanol. Mix by inverting the tube several times. Incubate at -20C for 10 min.
13. Centrifuge for 5 min at 14,000 rpm. Discard supernatant.
14. Rinse with 75% EtOH. Dry briefly in vacuum desiccator.
15. Resuspend pellet in 150 ul water. Add 20 ul of 1M MgCl2 and incubate on ice for more than 30 minutes.
16. centrifuge for 5 minutes at 14,000 rpm. Pour supernatent into a new 1.5 ml tube. Add 10 ul of 3M soduim acetate, ph 7.0
17. Add 400 ul cold EtOH. Centrifuge for 5 min at 14,000 rpm.
18. Discard the superantant solution. Rinse pellet with 75% EtOH. Dry briefly in vacuum desiccator.
19. Dissolve precipitate in 10 ul TE + RNase (1 mg/ml).
20. Use 5 ul for restriction digest.

Solution I: 50 mM glucose, 25 mM Tris ph 8.0, 10 mM EDTA ph 8.0
Solution II: 0.2M NaOH, 1% SDS

-smu2-

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