Problem of linker ligation to vector

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Yoshi
Yoshi's picture
Problem of linker ligation to vector

Now I need anyone's help, regarding linker ligation to vector, whichI tried several times in vain.
In order to create EcoRI site just before NcoI site of Promega pGL4 vector,
I ordered two combinations of linker oligos as follows. 

 
EcoRI-SalI-NcoI linker sense: 5'-catgcGAATTCGTCGAC-3'
 
 
 
EcoRI-SalI-NcoI  linker antisense: 5'-catgGTCGACGAATTCg-3'
 
 
EcoRI-BamHI-SalI-NcoI linker sense: 5'-catgcGAATTCGGATCCGTCGAC-3'
 
 
 
EcoRI-BamHI-SalI-NcoI linker antisense: 5'-catgGTCGACGGATCCGAATTCg-3'
 
BamHI and SalI sequences are not essential, but I included them considering possible enhancement of hybridization between sense and antisense oligos.
I made the 100 uM TE solution of the oligos.
To make sense and antisense oligos hybrize to each other, I mixed the same volume (10 ul each) and heated them to 99 ? for 5 min, then left them to room temperature for a few hours.
I linearized the pGL4 vector by NcoI, CIAP-treated and comfirmed the band by gel electrophoresis.
Ligation was performed using 100 ng (?0.03 pmol) of pGL/NcoI+CIAP and 50 ng hybridized oligos (?5 pmol), using NEB T4 ligase.
As a control, I prepared reaction without oligos.
Colony number is almost identical and low (around 100 per plate) regardless of the presence or absence of oligos.
I checked dozens of colonies but none had EcoRI site.
How can I solve the problem? 
WIthout this step successful, I cannot proceed to the next step. Please give me advices.
Thank you in advance. 
Best regards, 
Yoshi

RLS
RLS's picture
 I need a little more

 I need a little more information; did you phosphorylate the oligos with T4 PNK prior to ligating into the vector? Oligos are not generally phosphorylated.

Yoshi
Yoshi's picture
That's right!

That's right!

Reviewing my procedures, I have just noticed that my oligos were not phosphorylated!
Thank you very much for your message.
Yoshi

Yoshi
Yoshi's picture
One more question,

One more question,
Regarding the phosphorylation of oligos, I have no experience.
Long time ago, I used a premade phosphorylated oligos as a linker.
Would you teach me how to phosphorylate oligos at 5' end and purlfy the oligos for linker ligation?
Best regards,
Yoshi

pguo
pguo's picture
(Source:  http://www.e-biotek

(Source:  http://www.e-biotek.com/dna/141-dna-ligation-protocol-.html )
Abstract
This is a consensus protocol. See the bottom of this article for specific protocols.
DNA ligation is the process of joining together two DNA molecule ends (either from the same or different molecules). Specifically, it involves creating a phosphodiester bond bond between the 3' hydroxyl of one nucleotide and the 5' phosphate of another. This reaction is usually catalyzed by a DNA ligase enzyme. This enzyme will ligate DNA fragments having blunt or overhanging, complementary, 'sticky' ends. Typically, it is easier to ligate molecules with complementary sticky ends than blunt ends. T4 DNA ligase is the most commonly used DNA ligase for molecular biology techniques and can ligate 'sticky' or blunt ends.
 
The two components of the DNA in the ligation reaction should be equimolar and around 100μg/ml. Most commonly, one wants to ligate an insert DNA molecule into a plasmid, ready for bacterial transformation. Typically, DNA and plasmid vector are individually cut to yield complementary ends, then both are added to a ligation reaction to be circularised by DNA ligase. If the plasmid backbone to insert DNA ratio is too high then excess 'empty' mono and polymeric plasmids will be generated. If the ratio is too low then the result may be an excess of linear and circular homo- and heteropolymers.
 
Materials
 
Reagents
T4 DNA ligase
10x T4 DNA Ligase Buffer
Deionized, sterile H2O
Purified, linearized vector (likely in H2O or EB)
Purified, linearized insert (likely in H2O or EB)
 
Equipment
 
Vortex
 
Procedure
10μL Ligation Mix
Larger ligation mixes are also commonly used
1.0 μL 10X T4 ligase buffer
6:1 molar ratio of insert to vector (~10ng vector)
Add (8.5 - vector and insert volume)μl ddH2O
0.5 μL T4 Ligase
Calculating Insert Amount
Insert Mass in ng=6 x (insert length in bp / vector length in bp) x vector mass in ng
The insert to vector molar ratio can have a significant effect on the outcome of a ligation and subsequent transformation step. Molar ratios can vary from a 1:1 insert to vector molar ratio to 10:1. It may be necessary to try several ratios in parallel for best results.
 

Method
 
Add appropriate amount of deionized H2O to sterile 0.6 mL tube
Add 1 μL ligation buffer to the tube.
Vortex buffer before pipetting to ensure that it is well-mixed.
Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation.
Add appropriate amount of insert to the tube.
Add appropriate amount of vector to the tube.
Add 0.5 μL ligase.
Vortex ligase before pipetting to ensure that it is well-mixed.
Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 0.5 μL, just touch your tip to the surface of the liquid when pipetting.
Let the 10 μL solution sit at 22.5°C for 30 mins
Denature the ligase at 65°C for 10min
Dialyze for 20 minutes if electroporating
Use disks shiny side up
Store at -20°C
Critical steps
 
Troubleshooting
 
Factors affecting efficiency
From Tom Ellis
A protocol analysis experiment for a typical DNA ligation (7.2 kb vector + 0.6 kb insert, sticky ends) gave optimal ligation efficiency when 50 ng of vector was ligated overnight at 16°C with a 2:1 insert:vector molar ratio and standard T4 ligase. Ligase was heat inactivated at 65°C for 20 mins before 2 μL (of 20 μL) was used to transform commercial heat-shock competent cells.
Ligation efficiency was marginally decreased by
Doing a 1 hr ligation at room temperature
Using 100 ng vector
Using insert:vector molar ratios of 5:1 and 1:1
Ligation efficiency was noticably decreased (x100) by
Sticky end ligation with a larger insert (5.2 kb vector + 2.6 kb insert)
Blunt end ligation
Ligation efficiency was severely decreased (x10000) by
Using DNA fragments that have been exposed to ethidium bromide and UV during the gel extraction procedure (difficult to avoid but heartily recommended)
Using the NEB Quick Ligation Kit (possibly a bad batch)
For additional troublshooting, check out the NEB FAQ page for T4 ligation:
 
Notes
 
Make sure the buffer is completely melted and dissolved. The white precipitate is BSA according to NEB. Make sure the buffer still smells strongly like "wet dog" (to check if the DTT is still good).
Because ligase buffer contains ATP, which is unstable and degraded by multiple freeze/thaw cycles, you may want to make 10-20ul aliquots from the original tube. Ligase buffer may be spiked with additional ATP.
If you are having trouble with your ligation, NEB offers FAQ's (Quick Ligation T4 DNA ligase) and tips to help.
Prior to the ligation, some heat their DNA slightly (maybe ~37°C) to melt any sticky ends which may have annealed improperly at low temperatures.
Tom Knight has read that ligase can inhibit transformation. By heat-inactivating the ligase, this inhibition can be avoided. However, according to the NEB FAQ, heat-inactivation of PEG (which is present in the ligation reaction) also inhibits transformation, therefore a spin-column purification is recommended prior to transformation if you are having problems.
Treating PCR products with proteinase K prior to restriction digest dramatically improves the efficiency of subsequent ligation reactions. [1]
Using SYBR Safe DNA Gel Stain is a safer, non-carcinogenic alternative to ethidium bromide.
T4 DNA Ligase is very sensitive to shear, so spinning your ligation mix or vortexing it to mix it can affect your yields. Instead try mixing with the pipette tip or slowly resuspending the solution.

 
Need more information about DNA and vector ligation, please visit: http://www.e-biotek.com

R Bishop
R Bishop's picture
Phosphorylation of oligos is

Phosphorylation of oligos is pretty simple.  You can use any commercially available T4 DNA Kinase.

1. Mix 10µl of each oligo (1mg/ml) and put them in a ~100°C in a heat block. Immediately turn off the heat block and let the oligos cool down slowly (I use a PCR machine).
2. Then the phosphorylation reaction is
20µl oligos
5µl 10x Kinase buffer
10µl 10mM ATP
10µl T4 polynucleotide kinase
5µl dH2O
Incubate at 37°C for 1hr
3. 70°C for 10 min 9 (kills the kinase) and proceed to your ligation reaction.
 
 
Rus

Yoshi
Yoshi's picture
 Dear Peter and R Bishop,

 Dear Peter and R Bishop,
 
Thank you for your helpful advice.
Now I found my reason for the failure and am relieved 
Yoshi
 

Jason King
Jason King's picture
Wouldn't it be cheaper to re

Wouldn't it be cheaper to re-order the primers?

RLS
RLS's picture
It's likely not to cheaper to

It's likely not to cheaper to reorder primers with a 5' phosphate; check with the company from which you order oligos. I routinely order the oligos without modification because adding modifications is typically quite expensive and phosphorylate myself; it's really quite easy to do, as R. Bishop's protocol demonstrates.  

RLS
RLS's picture
 I have posted a protocol for

 I have posted a protocol for cloning tRNA genes into a vector starting with oligos, which includes both hybridization and phosphorylation of the oligos: http://www.scientistsolutions.com/science-protocols_displayprotocol_78e555ed-2f11-41fd-869c-622090c425d2_7939f6c3-7526-4f0f-81e7-33b7eaaeedbd.aspx
The same methods should also work for your hybridization and phosphorylation of the linker you are trying to insert.

Yoshi
Yoshi's picture
 Thank you for the detail

 Thank you for the detail protocol.
This time, I have reordered phosphorylated oligos.
One more simple question.
Usually I make a oligo dissolve in TE. In order to make sense and antisense oligos hybridize, should I dissolve them in deionized water? 
Or is TE OK?
 
 

R Bishop
R Bishop's picture
TE should be fine, but I

TE should be fine, but I usually use diH2O.

PhilM
PhilM's picture
TE should be fine, and when

TE should be fine, and when you add the oligos to a ligation mix it should be sufficiently diluted that the EDTA does not inhibit any reactoins, but to be safe you can dissolve DNA/oligos in Tris buffer pH 8.  This is exactly the same as TE buffer except without the EDTA (the "E"). 
The EDTA is included in buffers to stabize the DNA for long term storage.

Yoshi
Yoshi's picture
 Thanks!  Today I will try

 Thanks!  Today I will try ligation and transformation.
Yoshi

qiang wang
qiang wang's picture
PhilM wrote:

PhilM wrote:

TE should be fine, and when you add the oligos to a ligation mix it should be sufficiently diluted that the EDTA does not inhibit any reactoins, but to be safe you can dissolve DNA/oligos in Tris buffer pH 8.  This is exactly the same as TE buffer except without the EDTA (the "E"). 
The EDTA is included in buffers to stabize the DNA for long term storage.

 
I always use Tris-H2O (adijust ddH2O with 1M Tris pH8.0, to pH8.0), wich makes DNA/RNA dissolve efficiently, and no worrys about EDTA

RLS
RLS's picture
 Hi Yoshi,

 Hi Yoshi,
 
Were you able to get the cloning to work?

Yoshi
Yoshi's picture
 Dear RLS and all,

 Dear RLS and all,
 
 Yes, this time the colony number with phospho-oligo insert was much more than that without the insert  as I expected.
I checked the sequences and some clone have multiple inserts. I finally isolated the desired one with single insert and EcoRI-NcoI orientation.
Now I am proceeding to the next step.
 
Thank you all for a lot of advices!
Also sorry for me to be late to report my results.
Yoshi

Yoshi
Yoshi's picture
Hi all,

Hi all,

I have made a new construct in which IRES sequence was placed in front of Luciferase in pGL4. When I used the SV40 enhancer/promoter-driven IRES-luciferase in the pGL4 as a positive control, lucifearase activity was detected, although the activity of luciferase after IRES was less by 5 fold than that without IRES. Still the activity was more than that of mock (promoterless pGL4). Then I used a promoter of my interest and the luciferase activity was high enough without IRES. But when I inserted IRES in front of luciferase, the activity is almost negative that is the same level of promoterless construct (without IRES also). I know that IRES decrease the expression of the gene placed after IRES.

So now I am considering the alternative way to enhance the luciferase activity. I am wondering whether intronic sequence between promoter and IRES might enhance the expression of luciferase.

Does anyone give an advice and comments?
Thanks in advance.
Yoshi

dav0082
dav0082's picture
 Linker ligation - Vector

 Linker ligation - Vector size 7.5 kb double digested to get 6.9 kb size of interest. 
I want to ligate 50 bp linker digested with same RE's as that of vector to yield approx. 30bp linker for ligation.linker was made by hybridizing two oligos (50bp) 
Does this need phosphorylation of linker after cutting with RE's 
Vector is CIP treated.