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Need advice on cloning very small insert into a vector


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#1 stagius24

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Posted 02 September 2010 - 09:34 PM

Hello,

This is the first time I approach cloning. I am trying to clone a very small DNA fragment, 66bp, which I ordered as single strain oligonucleotide with RE site, phosphorylated and anneal them together.
I haven't done anything yet, still seeking for the best method. Here is my plan so far.

Vector: 5kb, GST tag, low copy plasmid
Supplied vector is 100ng/ul. I will cut it with XhoI ( 5ul DNA ~500ng, 2ul buffer 3, .2ul BSA, 1ul XhoI, water in a total of 20ul), and incubate for 2 hours at 37C, and do a heat inactivation at 65C for 20min.

Next, I will use CIP to dephosphorylate the vector ( 20ul from above rxn + .5ul CIP in 1 hours at 37C). Run 2ul on gel to confirm linearity.

Finally, using Qiagen pcr purification kit to get rid of all enzymatic, re-suspend DNA in 20ul water. The amount of DNA at this stage is roughly 500ng. At this stage, I think the vector is ready for ligation. Am I missing something? I will use 3ul of solution at this stage in the ligation reaction with the 66bp insert.

Insert 66bp plus RE site for XhoI and SalI, phosphorylated
Since this is a small insert, I am worry about the ligation reaction. How much ng of insert do I need? 3:1 or 5:1 ratio? Is it considered a difficult ligation that need overnight ligation?

This is my plan so far. Please give me feedback, and tips. I would rather spent more time research and do it right than do it again and again.

Regards,

#2 phage434

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Posted 03 September 2010 - 04:59 AM

Personally, I would avoid CIP (and attempt to avoid dephosphorylation entirely). If you need dephosphorylation, using shrimp alkaline phosphatase or antarctic phosphatase would certainly be a better choice. Avoid doing this reaction too long. An hour is probably too long.

SalI has a very bad reputation for cutting linear DNA fragments near their ends. Can you substitute another enzyme?

You need to anneal your oligos. The easy way to do this is heat a beaker of water to 90 or so, put your oligos (equimolar) in a tube and put the tube in the water, letting the water cool down on the bench. You may need to do this in the presence of some salt.

You didn't say how you had designed the oligos. Do they already have the restriction digest overhangs in place, or do you need to cut them prior to insertion? If you are cutting them, you do not need to phosphorylate them. If you are annealing to produce the correct overhangs, then you do.

Your insert should be added to the ligation at near equimolar amounts as the vector. This means very small amounts of insert, since it is so short.

Have you considered how you will select for the correct results?

#3 stagius24

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Posted 03 September 2010 - 10:50 AM

Hello Phage434,

I am not worry about SalI being a bad cutter, since my oligos already have the RE site overhand. I just need to anneal them, and they are good to go.I only need to cut the supercoiled plasmid with XhoI.

Can you clarify more on the molar ratio of vector:insert? From what you say, equimolar is 1:1 ratio, right?

Noticed my plan doesnt used the gel extraction strategy to purify the plasmid from enzymes. After digestion and CIP, I only use Qiagen to purify the DNA. I dont know if I can substitue this for gel extraction. Someone in my lab suggested me to do a glassmilk method to extract the plasmid from the gel, but I figured that would take to long.

#4 Deepak Anand

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Posted 09 April 2012 - 05:59 AM

Hey,
I have done the same cloning of 66 pb DNA fragment which I have ordered as two complementary oligos already designed RE site. I dissolved the oligos in water and did annealing as recommended by Sigma protocol. ( http://www.sigmaaldr...ing-oligos.html)
You do not need to go for PNK reaction to add P at 5' end these oligos. ( you should not, so that they will never ligate together) If you have done that then don't use that. Use the double digested vector with these oligos and do ligation with what ever ratio, you will have more than 90 % positive clone for sure. For successful cloning First digest the vector with one Enzyme and gel elute the linear band. now do second digestion with double the amount of second Enzyme and gel elute again. before going for ligation check the DNA on gel. set up the ligation [with t4 DNA ligase Fermentas or Quck ligase NEB] and next day you have your clone.
(we do it on regular basis)
Good Luck




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