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yet one more cloning problem


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#1 Givi

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Posted 29 May 2009 - 12:15 PM

Dear friends, I have faced strange problem for which I don't have any explanations and hence no solutions.
background is following:
1) vector is digested by Nco I/ Hind III for 1 hour, than alkaline phosphatase is added and incubated for 15 min. digested vector (5400 b.p.) is purified from gel. between restriction sites there is an insert, so it is easy to detect that vector perfectly and completely digested.
2) two inserts are used. They are coding for same protein; however, one is 1200 b.p., another - 1218 bp, hence coding for a bit longer variant of protein.
Three ligation reactions were made: two for vector:insert and one with digested vector alone as control. Quick ligase from NEB was used accordingly to manufacturers recommendations.
Results:
after transformation and overninght cultivation on control plate (with empty vector) two colonies appeared; on plate with insert no.1 (1200 bp) five colonies appeared; on plate with insert no.2 >50 colonies appeared. For insert no.1 all 5 colonies were tested and all of them contained vector with insert; for insert no.2 (1218 bp) 9 colonies from plates were tested and none of them contained vector with insert, but empty vector (accordingly to size). When I say "colonies were tested" this means that overnight culture was made from a single colony, plasmid was purified with QIAGEN kit and digested with same NcoI/Hind III enzymes.
For transformation DH5 alfa cell were used and these cells were from the same tube (!) so no contamination should be connected with cells. Digested and dephosphorylated vector is the same in all three reactions, because single aliquot after purification was simply divided into three equal parts.
It seems that in one case cells somehow have "thrown" away my insert out of the vector. How it can be and how I can prevent this?

sincerelly hope for your advice

#2 T C

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Posted 31 May 2009 - 09:00 PM

Hey,

1. Its all fine, just repeat with the second cloning and you will get it.

2. Increase the time for phosphatase treatment...I think 15 minutes is too less. I generally incubate for 1 hour.

Best,
TC

#3 Givi

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Posted 01 June 2009 - 03:09 AM

Hey,

1. Its all fine, just repeat with the second cloning and you will get it.

2. Increase the time for phosphatase treatment...I think 15 minutes is too less. I generally incubate for 1 hour.

Best,
TC

1)I'm trying to be very careful with phosphatase treatment in order not to destroy my sticky ends, because I have read that it can happen.
2)I have checked my primers with "oligo calc" and have discovered self-annealing sites. What kind of problems these can cause and how to avoid such problems?

#4 phage434

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Posted 01 June 2009 - 03:46 AM

Might the second insert have been exposed to uv for relatively longer times? You have said nothing about the preparation of the inserts. If they were pcr'd, how were they purified? Were overhangs on the restriction sites long enough? How do you know they were cut effectively?

#5 Givi

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Posted 01 June 2009 - 08:30 AM

Might the second insert have been exposed to uv for relatively longer times? You have said nothing about the preparation of the inserts. If they were pcr'd, how were they purified? Were overhangs on the restriction sites long enough? How do you know they were cut effectively?


Usually I proceed my inserts in following way:
1) I make a PCR, afer I use 5 microliter of mix to check on a gel if PCR worked and if there is a band of appropriate size; usually I have only one band and it's o.k.
2) after checking, I purify the rest of mix using QIAGEN kit accordingly to their instructions
3) checking conc. with Nanodrop, making digestion (NEbuffer 2, NcoI/ Hind III).
4) to avoid exposure to UV, after digestion I'm again purifying insert with same pcr-purification kit, checking concentration with Nanodrop again
5) my vector has already an insert between Nco I and Hind III sites, so I can see that it was cut properly; vector I'm purifying from agarose gel;
6) every insert has 6 b.p. more after restriction site to be sure that enzyme will cut it properly.
for inert digestion I'm using same enzymes that for vector, so they should cut, however tomorrow I'll try to make control of insert digestion and ligation, as you suggested. But if these are not cut effectively, I don't know what I can change this.

#6 Givi

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Posted 03 June 2009 - 05:59 AM

Recently I have made a test of inserts. I had solutions of inserts, double digested with NcoI/Hind III. I decided to add ligase and check if I will see smear or a ladder of different sizes.
As control I have used non-ligated digested inserts.
So control (1200 bp) gives band between 1000 bp and 1500 bp which seems o be o.k.
re-ligated insert no.1. gives a single smeary band approx. at 1500 bp (accordingly to marker- 1 kb ladder from NEB).
re-ligated insert no.2 gives smeary band approx. at 1500 bp (accordingly to marker) and a smeary band at 3000 bp (acc. to marker) which should correspond to dimer.

I REALLY need some help in interpreting my weird result and solving the problems.

#7 phage434

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Posted 03 June 2009 - 05:07 PM

1. To get clearer bands, heat kill the ligase before loading the gel
2. The 1.5 Kb band indicates that nothing is ligating, so you have a problem with the digestion of both ends of your insert. Buffer conditions, insufficient overhangs, bad enzymes, bad cleanup after the reaction?
3. The 3 Kb band indicates that at least one end of your insert is ligating. If you take that product and digest with each of your enzymes independently, then you can figure out which of the enzymes is cutting and religating, and which is not. The enzyme that cuts the dimer is the one that is working (of course). What you want to have happen is for this cutting to give a double length band for both enzymes, which would indicate that both enzymes were working and ligating. The ratio of single to double length fragments on this cutting test give a direct measure of the effectiveness of your cutting and ligation efficiency.

#8 Givi

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Posted 04 June 2009 - 03:53 AM

Thank you for reply!
I'm making this kind of control at the moment. I just want to know one more general thing. As I have already written, I am purifying digested inserts with QIAGEn pcr purification kit, not from agarose gel(it is faster and insert is not exposed to UV). Can this affect somehow "sticky ends" of my insert?




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