11 replies to this topic

[sanguinara]

Hey pplz,

I was trying to cut a piece out of an existing plasmid and replace it with a promoter of my own. However, the RE sites on the plasmid are limited. The only thing that will cut at the 5' end is AfeI, which happens to be blunt. For the 3' end I picked BamHI.

Then I digested the plasmid with those 2 enzymes overnight, PCR'ed and digested the promoters with the corresponding REs. I also purified both digestion products using gel extraction. The vector end up being around 7kb while the insert is around 2kb. However after ligation and transformation, no colony appeared. I'm pretty sure the transformation is working since positive controls all look normal.

I tried both the Fermentas CloneJet and T4 DNA ligase for the ligation. I used 25ng of vector and 75ng of insert and ligated at 14 degrees for up to 24 hours or at room temperature for 1 hour. Neither strategy worked. Then I doubled the ligase concentration but still saw no colonies.

Right now I'm back to square one: is it even possible to ligate blunt and sticky ends at the same time? Should I just blunt down the BamHI site with Klenow and do a blunt ligation instead? Or would it be more time-efficient resorting to some other method (such as PCR fusion)?

Any hint would be helpful!! Thanks in advance~!  

Edited by sanguinara, 30 July 2010 - 02:33 PM.

[perneseblue]

sanguinara, on 30 July 2010 - 02:32 PM, said:

Hey pplz,

I was trying to cut a piece out of an existing plasmid and replace it with a promoter of my own. However, the RE sites on the plasmid are limited. The only thing that will cut at the 5' end is AfeI, which happens to be blunt. For the 3' end I picked BamHI.

Then I digested the plasmid with those 2 enzymes overnight, PCR'ed and digested the promoters with the corresponding REs. I also purified both digestion products using gel extraction. The vector end up being around 7kb while the insert is around 2kb. However after ligation and transformation, no colony appeared. I'm pretty sure the transformation is working since positive controls all look normal.

I tried both the Fermentas CloneJet and T4 DNA ligase for the ligation. I used 25ng of vector and 75ng of insert and ligated at 14 degrees for up to 24 hours or at room temperature for 1 hour. Neither strategy worked. Then I doubled the ligase concentration but still saw no colonies.

Right now I'm back to square one: is it even possible to ligate blunt and sticky ends at the same time? Should I just blunt down the BamHI site with Klenow and do a blunt ligation instead? Or would it be more time-efficient resorting to some other method (such as PCR fusion)?

Any hint would be helpful!! Thanks in advance~!  

1 - Yes, you can ligate a DNA fragments where one end is a blunt end and the other a staggered end. A double blunt end ligation is alot more harder to ligate than a blunt-staggered end ligation

2. How long did you digest the vector and insert for?

3- How many bp was added behind the restriction site?

[sanguinara]

@perneseblue
2. I digested both of them overnight.

3. 6bp spacers were added... Is that enough?

[perneseblue]

sanguinara, on 31 July 2010 - 01:06 PM, said:

@perneseblue
2. I digested both of them overnight.

3. 6bp spacers were added... Is that enough?

The DNA fragments should be digested. 6bp is enough. I don't see any major errors with your general description. The problem is probably in the detail

Did you dephosphorylate the vector?

Check to see that the enzymes BamHI and AfeI can individually linearise the plasmid. There might be a small chance that the enzyme has gone bad (very unlikely.. but you never know..I have seen vials of enzymes 15 years old in freezers)

Could you write out the digestion formulation that was used? The total volume of enzyme used should not exceed 5% of the total volume of the digest. Restriction enzymes comes in a glycerol storage buffer. While glycerol protects the enzyme it also interferes with its activity.

T4 DNA ligase and its buffer goes off easily. Dead T4 ligase is often the cause for a ligation failure when everything appears to be okay

The t4 ligase buffer has a strong smell. If the buffer does not smell, the buffer is dead. Bin and get new buffer. Always aliquot out new vials of T4 ligase buffer into smaller aliquotes. Do a test ligation . Ligate a sample of digest dna. Run the ligation mix on a gel. If you see the emergence of high molecular weight bands, the ligase is working. If you see no difference, the ligase is dead. Bin and buy a new one. Only buy small vials. A lab can rarely finish a big vial before the enzyme goes bad. And T4 ligase will go bad even if it is kept on ice.

Alternative
There are alternative you can consider. How big is your vector? You can PCR amplify both vector and insert to add your desired restriction sites. Use a proof reading polymerase such as Phusion (NEB) , it will (very likely) get the job.done with little trouble.

[sanguinara]

perneseblue, on 31 July 2010 - 07:49 PM, said:

sanguinara, on 31 July 2010 - 01:06 PM, said:

@perneseblue
2. I digested both of them overnight.

3. 6bp spacers were added... Is that enough?

The DNA fragments should be digested. 6bp is enough. I don't see any major errors with your general description. The problem is probably in the detail

Did you dephosphorylate the vector?

Check to see that the enzymes BamHI and AfeI can individually linearise the plasmid. There might be a small chance that the enzyme has gone bad (very unlikely.. but you never know..I have seen vials of enzymes 15 years old in freezers)

Could you write out the digestion formulation that was used? The total volume of enzyme used should not exceed 5% of the total volume of the digest. Restriction enzymes comes in a glycerol storage buffer. While glycerol protects the enzyme it also interferes with its activity.

T4 DNA ligase and its buffer goes off easily. Dead T4 ligase is often the cause for a ligation failure when everything appears to be okay

The t4 ligase buffer has a strong smell. If the buffer does not smell, the buffer is dead. Bin and get new buffer. Always aliquot out new vials of T4 ligase buffer into smaller aliquotes. Do a test ligation . Ligate a sample of digest dna. Run the ligation mix on a gel. If you see the emergence of high molecular weight bands, the ligase is working. If you see no difference, the ligase is dead. Bin and buy a new one. Only buy small vials. A lab can rarely finish a big vial before the enzyme goes bad. And T4 ligase will go bad even if it is kept on ice.

Alternative
There are alternative you can consider. How big is your vector? You can PCR amplify both vector and insert to add your desired restriction sites. Use a proof reading polymerase such as Phusion (NEB) , it will (very likely) get the job.done with little trouble.

No I have not dephosphorylated my vector...I thought it was unnecessary after RE digest?

My digestion mix contains 1 microliter of each of the RE, 10 microliter of BSA, 5 microliters of NEB buffer 4, and 2 microliter DNA. dH2O was added to bump the volume to 50 microliter.

The RE have worked on control groups, so I'm pretty sure they are working. The T4 ligase was just purchased less than 1 week ago, so I'm kinda assuming it's working as well. I'll definately sniff the buffer on Monday to make sure it's working.

On the other hand,my vector is around 7kb while the insert is 2kb. Right now I'm looking into PCR site-directed mutagenesis too in case ligation does not work.

And thanks so much for helping!!

[perneseblue]

sanguinara, on 01 August 2010 - 08:14 AM, said:

No I have not dephosphorylated my vector...I thought it was unnecessary after RE digest?

My digestion mix contains 1 microliter of each of the RE, 10 microliter of BSA, 5 microliters of NEB buffer 4, and 2 microliter DNA. dH2O was added to bump the volume to 50 microliter.

The RE have worked on control groups, so I'm pretty sure they are working. The T4 ligase was just purchased less than 1 week ago, so I'm kinda assuming it's working as well. I'll definately sniff the buffer on Monday to make sure it's working.

On the other hand,my vector is around 7kb while the insert is 2kb. Right now I'm looking into PCR site-directed mutagenesis too in case ligation does not work.

And thanks so much for helping!!

You are correct. It is unnecessary  to dephos in a situation where the ends of the vector are non-compatible. So this is not a problem with CIP.

Are you using NEB 100X BSA? If so, that is a lot of BSA in the digest. Has such a digest formulation worked before?  I am uncertain if the digest will be okay with so much BSA.

I would suggest doing a digest just to see that the RE enzyme are working. Just to put to an end that concern.

[sanguinara]

perneseblue, on 01 August 2010 - 05:04 PM, said:

sanguinara, on 01 August 2010 - 08:14 AM, said:

No I have not dephosphorylated my vector...I thought it was unnecessary after RE digest?

My digestion mix contains 1 microliter of each of the RE, 10 microliter of BSA, 5 microliters of NEB buffer 4, and 2 microliter DNA. dH2O was added to bump the volume to 50 microliter.

The RE have worked on control groups, so I'm pretty sure they are working. The T4 ligase was just purchased less than 1 week ago, so I'm kinda assuming it's working as well. I'll definately sniff the buffer on Monday to make sure it's working.

On the other hand,my vector is around 7kb while the insert is 2kb. Right now I'm looking into PCR site-directed mutagenesis too in case ligation does not work.

And thanks so much for helping!!

You are correct. It is unnecessary  to dephos in a situation where the ends of the vector are non-compatible. So this is not a problem with CIP.

Are you using NEB 100X BSA? If so, that is a lot of BSA in the digest. Has such a digest formulation worked before?  I am uncertain if the digest will be okay with so much BSA.

I would suggest doing a digest just to see that the RE enzyme are working. Just to put to an end that concern.

Oops my bad! The BSA is indeed 100x! It's amazing that it actually worked before! I'll re-digest my stuff anyways just to be sure...Thanks!!!

[sanguinara]

Case update:

I have re-digested the plasmid and PCR product, cleaned them up using gel extraction and did several trials of ligations. (I'm sure that the plasmid has been linearized this time.) I used various concentration of insert and vector, tried multiple kits but still got no product. There were some colonies on the plates but they all look weird (smaller and whiter than usual) and do not grow in liquid medium. I guess they were negatives?

Right now I'm working with Fermentas' Rapid Ligation kit, hoping for a miracle . Supervisor said to try phenol/chloroform extraction and use fresh PCR product so I'm gonna try those as well.... Any comments/suggestions will be helpful!! Thanks~!!!!!!

[NemomeN007]

Well...it's a long shot, but without a map and sequence, there is a chance that you're removing an important part of the backbone..either part of the ori or part of the resistance marker/promoter region which will result in no colonies after transformation....

[perneseblue]

If the insert and vector were gell purified, there is no need for phenol-extract step. The DNA is clean. If you do phenol-chloroform extract, ethanol precipitate the DNA, and wash the DNA pellet in 70% ethanol. Phenol can inhibit down stream enzymatic reactions.

If the 1-blunt end/1-stick end ligation strategy does not work, you might want to consider a different strategy. Perhaps consider PCRing the vector to add your desired restriction site. Both vector and insert can be modified by PCR.

[sanguinara]

NemomeN007, on 10 August 2010 - 07:27 PM, said:

Well...it's a long shot, but without a map and sequence, there is a chance that you're removing an important part of the backbone..either part of the ori or part of the resistance marker/promoter region which will result in no colonies after transformation....

Thanks for the input! I do have a (partial) map so I'm partially confident that no important parts are cut out.

[sanguinara]

perneseblue, on 11 August 2010 - 08:13 PM, said:

If the insert and vector were gell purified, there is no need for phenol-extract step. The DNA is clean. If you do phenol-chloroform extract, ethanol precipitate the DNA, and wash the DNA pellet in 70% ethanol. Phenol can inhibit down stream enzymatic reactions.

If the 1-blunt end/1-stick end ligation strategy does not work, you might want to consider a different strategy. Perhaps consider PCRing the vector to add your desired restriction site. Both vector and insert can be modified by PCR.

I'm on it! I managed to ligate the insert into a Clonejet vector after blunting it. Hmm. I'll work on the vector now.

Thank~~!!