Clonierungsstrategie- hat jemand eine Idee ? |
| brand | 2007-10-11 09:53:08 |
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Beitrag #1 • | Dear all,
my aim is to subclone a protein of 6 kbp into pcDNA3-vector (5,4 kbp). To obtain this construct I used the following strategies: (1) subcloning the full length protein into the pcDNA3-vector (not successful), (2) first cutting the protein into two fragments (both about 3 kbp long) with the aim to subclone them successivelyinto the vector: I first sucloned the first part of the protein into pcDNA3 successfully. Then I was trying to subclone the second part of the protein into this construct which was not successful. For ligation I used different ligases and different strategies: Quick-Ligation Kit, overnight incubation with T4 DNA Ligase, heating up the fragments for 5 min before ligation, adding DMSO to the ligation assay.
Does anyone has an idea which other strategies could be used to obtain the above mentioned construct ?
| | acribio | 2007-10-11 15:58:26 |
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Beitrag #2 • | Dear Mr. Brand!
First of all: Welcome to this forum!!
In my humble opinion, the cloning of large insert fragments into large vectors is a hard and long work with finally NO result! ;)
I have seen quite a lot of scientists who needed several months (in fact YEARS) to accomplish this task!
Even I needed weeks! :D
Okay I'm sure you have done quite a bit to achieve success, and thus I don't want to bother you with tips, which won't help you. Some hints I will give:
- Don't use two different restriction sites, the overall yield in the cleaning step is too low
- Sticky ends (mostly 4 bp overhang) have a too small affinity, so IF... the ends of vector and insert will find
they will not stay together too long, until the ligase will come around...
In one word: Your task is really hard! The classic way of cloning is voodoo-like, and you need a lot of luck.
| | acribio | 2007-10-11 16:25:22 |
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Beitrag #3 • | I will give you now an interesting different approach, which might be very easy and fast, though I have only experience with this method with a bacterial vector and a short insert (all in all 2.5kbp).
I have to appologise in advance for my poor drawings :-)
My recommendation: Give PCR-cloning a try.
I will explain the steps of the method. I made some cartoons to make things clearer.
| | acribio | 2007-10-11 16:34:36 |
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Beitrag #4 • | Pic. 1: Vector, Insert and two primers, which overlap the cloning region round about 21 bp on each strand
The vector is on to the left, the insert, which has the same size is on the right. The fragments are e.g. cleaned DNA from Agarose or size-exclusion-chromatography
Besides you see two small primers which are complementary to vector and insert. They should contain cloning sites, and may correct the reading frame, and and and...
| | acribio | 2007-10-11 16:40:40 |
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Beitrag #5 • | Pic. 2: 1st cycle of PCR
A PCR will complete the strands in the shown manner.
For this PCR you take the Pfu Polymerase or Pwo Polymerase, which are proofreading enzymes and will reduce the likelihood / probability of building in mistakes. (you need huge extension times for this! See below)
| | acribio | 2007-10-11 16:46:30 |
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Beitrag #6 • | Pic. 3: The first set of pcr products
One has to be careful, due to the fact that not every overhang will be completed by the polymerases. But finally you get both cutting sites completed... | | acribio | 2007-10-11 16:54:36 |
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Beitrag #7 • | Pic. 4: 2nd and further pcr cycles
In the next pcr cycles the long fragments will overlap with 21bp and the strands will be completed
| | acribio | 2007-10-11 16:56:16 |
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Beitrag #8 • | Pic. 5: The other strand vice versa
 | | acribio | 2007-10-11 17:10:00 |
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Beitrag #9 • | Pic. 6: The final step: Restriction digestion of the products
Now: What is the result of this PCR??
You will get a huge variety of different products! This is nearly waste. If you think that you can use this for cloning you are eighter stupid or clever :-) (may be even this works...)
To get a clonable construct a unique restriction digestion helps to get nice ligation product, which are easy to ligate!
The efficiency of ligation with only one cutting site ist extremly higher, than cloning with two cutting sites...
I tried this, and it worked very well with 2.5 kbp...
The PCR amplifcation works with such large vectors, I have experience with this. But!! : You need VERY long extension times, du to the fact, that proofreading enzymes are very slow!
For this example:
- round about 6kbp to amplify in each cycle
- I calculate 6bp/sec for pfu polymerase
6000bp / 6 bp/sec = 1000sec /cycle!
1000sec/cycle / 60sec/min = 17min !
but it works! Take 40 cycles and more, and you have lots of product!
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