How to ligate ds-oligos into a vector? - (Aug/25/2004 )
Hi,guys:
I try to ligase a ~40bps DNA into a single digested Vector.I got this ~40 bps DNA from annealing two complementary oligos, there are two same restriction enzyme sites in both sides of oligos. I didn't phosphorylate my oligos and also didn't dephosphorylate my vector.I use the big molar ration of insert DNA to digested Vector,try to kill reaction. But I didn't get any ligation product, only this digested vector ligase itself. some person has such experience not to phosphorylate and dephosphorylate vector,and got product,but I didn't succeed. do you have some suggestions? or I must do phorsphorylation and diphosphorylation?
appreciate your help!
Viki
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dear friend you do first phos`porylate your insert then dephporylate your vector and use higer molaer ratio of your insert like 1:5 take like 3 microliter vector and 15 your insert and use good competent cell and at the time of reaction mixture use 2 microliter of 10mM ATP also before doing chek your sequence also that it is toxic or not .
tell me two things....
1. was your vector digested with single or double Restriction enzyme...
2. have you seen prominent annealing of your oligos....
for better result phosphoryaltion of oligos is recommended.
try to avoid akaline phosphatase treatment.
best wishes
amit
Take 10uL of the annealed oligos, dilute in 990uL H2O. Use 2uL of this dilution for ligation.
my total primer size is 37 bp and so I guess I can skip the gel purification of the primers or can I do it using say Qiagen gel extraction kit?
also I have never used buffer E so I searched Promega and could only find buffer A, B, C and D for double digestion of DNA!!
when you say water bath do uou mean 70C
I have tried doing the technique you suggest but without the buffer E and putting the primers to anneal in a water bath bringing down temp by 10 degrees carrying on for the whole day long.
and at the end I got the colonies I said before!!
Thanks again
I wonder whether heating up already phosphorylated oligos to 95 °C during annealing could lead to some dephosphorylation... Any idea? thx
Hi, BadCell
I have same problem with cloning ds-oligos, I want to clone a 60-mer synthetic oligo with sticky end in BglI/PstI sites of pACYC177, as you know if it works I should get the KanR and AmpS colonies, I synthesized the ss-oligos and annealed them, and now I should have an ds-oligos with sticky ends (BglI/PstI), I would appreciate it if you tell me and step by step cloning procedures to clone it. Regards,
Farhad
I try to ligase a ~40bps DNA into a single digested Vector.I got this ~40 bps DNA from annealing two complementary oligos, there are two same restriction enzyme sites in both sides of oligos. I didn't phosphorylate my oligos and also didn't dephosphorylate my vector.I use the big molar ration of insert DNA to digested Vector,try to kill reaction. But I didn't get any ligation product, only this digested vector ligase itself. some person has such experience not to phosphorylate and dephosphorylate vector,and got product,but I didn't succeed. do you have some suggestions? or I must do phorsphorylation and diphosphorylation?
appreciate your help!
Viki
Has anyone tried concatenated the ds stranded oligo and then ligating into a vector? I am still on the concatenation step but my gel shows a smear rather than discrete bands surprisingly.
Hi zhgljj,
check this post http://www.protocol-online.org/forums/inde...?showtopic=6527
you can run a gel after annealing along side with single stranded oligos. You will find ds-oligos migrate differently from ss-oligos.
I ran a 2.5% gel to check my oligos (45 & 53 bases) and annealing. I saw the difference between 45 bases and 53 bases, but no difference between 53base oligo and annealed double-strand DNA. So, my annealing didn't work? I have tried several times.
My annealing : 1:1 molar ratio of the two oligos, concentration is about 3ug/ul for each, total volume was about 100ul.
I boiled water to 90C, put the annealing tube in. keep all things in a box to let them cool down slowly. Overnight.
BTW, the oligos were from idt, I dissolved them in dH2O.
Hi zhgljj,
check this post http://www.protocol-online.org/forums/inde...?showtopic=6527
you can run a gel after annealing along side with single stranded oligos. You will find ds-oligos migrate differently from ss-oligos.
I ran a 2.5% gel to check my oligos (45 & 53 bases) and annealing. I saw the difference between 45 bases and 53 bases, but no difference between 53base oligo and annealed double-strand DNA. So, my annealing didn't work? I have tried several times.
My annealing : 1:1 molar ratio of the two oligos, concentration is about 3ug/ul for each, total volume was about 100ul.
I boiled water to 90C, put the annealing tube in. keep all things in a box to let them cool down slowly. Overnight.
BTW, the oligos were from idt, I dissolved them in dH2O.
Our annealing recipe has 10 mM MgCl2, 50 mM NaCl, plus 20 mM Tris. IF you have any regions of potential secondary structure, the magnesium will fix it, and the salt is just generally good.
You definitely need salt in an annealing buffer, and the one swanny suggests looks good to me. Be careful of DNAse contamination, since this buffer is pretty good for making it active.
I read this thread before I attempted my annealing and ligation so thought i'd share my experience.
Two oligos added in equimolar amounts (both just in TE buffer),then put through the following programme on a Biorad DNA Engine cyler.
2 min 95C
2 min 72C
2 min 37C
2 min 25C
On ice until use (approx 1 hour when I did it). Oligos were designed to give an EcoRI and SpeI cloning site. Oligos were not phosphorylated.
Ligated as follows.
50ng vector (EcoRI/SpeI double digestion,not dephosphorylated,purified using a Qiagen maxtract column), 1X T4 DNA ligase buffer, 200U T4 DNA ligase (NEB), 0.3mg/ml BSA, 1ul annealed oligo (various ratios were used but volume kept to 1ul) then up to 15ul with ddwater. 3 hours at room temperature.
I then digested the ligation reaction with MluI as this site was inbetween the EcoRI and SpeI cloning sites in the MCS with 5units for 0.5 hours ( I must thank Badcell for that nice little hint ), which would get rid of any undigested vector.
Transformed 2ul as normal. 1:1 and 1:2 vector:insert molar ratio were the only ones to work and in all honest I only got a few colonies but you only need one !
Think that is all !
can any one tell me which type of DNA moves faster in a gel. linear one or the circular.
Also recently i cloned my gene in a vector. to confirm it i done gene release with double digestion. I then run gel with only vector on one lane and the digestion on other lane. I found that the double digested vector DNA in second lane is slighely above the control vector in the gel. what may be the reason... 