16 replies to this topic

[sou]

Hi evry1

I have a ligation problem. I am ligating a 300bp insert into a 12.5kb vector. The insert is a PCR product which is double digested for 4hrs with EcoRI/BamHI. The vector is also digested for 1 hour with the same enzymes and treated with SAP. After ligation i get more colonies on my control plate (only vector) than the insert+vector plate!! i have tried using a 1:6 insert:vector ratio, doing a blind ligation with 6ul digested vector and 1ul digested insert and lots of different things but the thing never works!!!!
Plz help!

[phage434]

Provide LOTS more detail on exactly what and how you are doing this.   How are you purifying the DNA after digestion? Is the ratio a molar ratio or a weight ratio? How do you do the ligation, exactly? How do you do the SAP digestion, exactly? How do you purify after that?

[sou]

phage434, on 17 October 2011 - 01:44 PM, said:

Provide LOTS more detail on exactly what and how you are doing this.   How are you purifying the DNA after digestion? Is the ratio a molar ratio or a weight ratio? How do you do the ligation, exactly? How do you do the SAP digestion, exactly? How do you purify after that?

The vector is run on gel after digestion and gel extracted with the quiagen kit. The insert after digestion is extracted using the purification kit from quiagen. The ratio used for ligation was the molar ratio. Ligation done using the T4 ligase from fermentas, overnight ligation at 16C. For the SAP digestion, SAP from fermentas was used. Its supposed to be active in the EcoRI buffer (thats what the website said!) So after the double digestion (EcoRI/BamHI in EcoRI buffer) of the vector, I used SAP in the same buffer, heat inactivated the mix, run on gel and extracted. I am fairly new to this field!

[almost a doctor]

Shouldn't the ratio be  1:6   VECTOR:insert, and not the way you have it.  Or is this a typo?
I've not done cloning in a long time, but I'm pretty sure you need more insert than vector. Think about it, your vector is 4x bigger than your insert, and you are putting 6x more....   I don't think the insert is getting a fair chance there but rather getting lost in the mix

Just my thoughts.

[sou]

almost a doctor, on 18 October 2011 - 01:20 AM, said:

Shouldn't the ratio be  1:6   VECTOR:insert, and not the way you have it.  Or is this a typo?
I've not done cloning in a long time, but I'm pretty sure you need more insert than vector. Think about it, your vector is 4x bigger than your insert, and you are putting 6x more....   I don't think the insert is getting a fair chance there but rather getting lost in the mix

Just my thoughts.

sorry! my mistake! it was 1:6 vector:insert ratio!

[GNANA]

after ligation rxn run some 2 microlitres on gel with vector alone as control, to make sure whether the ligation worked or not....

[sou]

GNANA, on 18 October 2011 - 05:30 AM, said:

after ligation rxn run some 2 microlitres on gel with vector alone as control, to make sure whether the ligation worked or not....

well I use just around 50ng of vector for the ligation reaction. taking 2uL from a ligation reaction of 20ul in which the total plasmid is 50ng, will anything be visible on the gel???

[GNANA]

i usually do 10 microlitre ligation rxn,said in that sense...i dont think you would tranform all the 20 microlitres, or run some 5 microlitres you shd able to visualize....

[sou]

GNANA, on 18 October 2011 - 07:08 AM, said:

i usually do 10 microlitre ligation rxn,said in that sense...i dont think you would tranform all the 20 microlitres, or run some 5 microlitres you shd able to visualize....

I use 10uL of the ligation mix to transform 100uL of dh5a. but I will try this suggestion with the 5ul running.. thx! One question here.. some people suggest to use an enzyme which cuts the vector if the vector has self ligated or if the insert is not there (or something in that sense! ) the advantage of this method being, before transformation I'm eliminating all the false positives.. so if I am using such an enzyme to cut the ligation mix, can I do the digestion in the same buffer? I mean wouldn't the DTT in the ligation buffer in any way inhibit the enzyme?
In between, ru from India?

[sou]

OK people Im officially fedup with this cloning! it just doesnt want to work!
Below is a detailed description of what I am trying to do here.

My plasmid is 12.5kb I am cutting it with SmaI, 1 hour at 25C, SAP treating it for 1hr at 37C, heat inactivating the SAP at 70C for 15min, cool on bench. Then I heat the digested plasmid with loading dye & SDS solution at 65C (fermentas recomends this step - my SAP is from fermentas) cool on bench, keep on ice and run on gel. I post stain the gel with GRGreen and gel extract with qiagen kit. The elution is with MQ.

For my insert, it is amplified from the total RNA using the Invitrogen superscript RT PCR kit. Its 300bp in length. I treat the insert with kinase for 1 hour (trying to do a blunt end ligation, but my primers are not phosphorylated hence the kinase) heat inactivate the kinase. let it cool on bench. heat the mix with loading dye & SDS at 65 (again following fermentas!) cool on bench and keep on ice before running on gel. post stained with GRGreen. I see a tight band at 300bp gel extract with qiagen kit again and elute with MQ.

Now for the ligation - doing the ligation with 50ng plasmid at 1:6 vector:insert ratio. Ligase from fermentas, ligated at 22C for 3hrs, heat inactivated and cooled at RT. Transformed into 100uL competent cells (home made DH5a chemical competent cells) on ice for 15min, 42C for 1min and ice for 10mins. 1mL of LB added, incubated for 1hr at 37C. spin down, resuspend in 100uL LB, 50uL spread on selection plates.

these are the results that i have got thus far
- no colonies

what am i doing wrong here??????
I once used all of the ligation mix (20uL) for the transformation into 100uL cells - no colonies
10ul ligation mix - no colonies
changed the competent cells, prepared a fresh stock - no colonies
did overnight ligation at 16C - no colonies

what am i doing wrong here??
HELP!!!!!

[almost a doctor]

A couple of questions that might seem obvious but just in case.

1. do you have a transformation control?  
2. do you have a ligation control?

If struggling with blunt end, could you not just redesign the primers to include RE and do directed cloning?   Your first post with this problem is from over a month ago, I think is time to change strategy. Cloning can be a pain in the b***  but should not take that long to work either  

[sou]

almost a doctor, on 02 December 2011 - 03:54 AM, said:

A couple of questions that might seem obvious but just in case.

1. do you have a transformation control?  
2. do you have a ligation control?

If struggling with blunt end, could you not just redesign the primers to include RE and do directed cloning?   Your first post with this problem is from over a month ago, I think is time to change strategy. Cloning can be a pain in the b***  but should not take that long to work either  

I have an intact plasmid at 150pg as transformation control and single digested plasmid as ligation control. n both give colonies.
we designed the primers for directed cloning with EcoRI/BamHI (the one month ago post!) which was not working (we put less of the extra bp at the ends - the enzyme was not cutting) hence planned the blunt ligation (instead of designing a new primer)
thought "blunt ligation - its a piece of cake!" STUPID ME! I am new to this cloning business!
anyway thx for the reply! but if u have any suggestion plz post!!! thx in advance!

[phage434]

When you transform 1 ul of your 150 pg/ul DNA, how many colonies do you get?  Why are you recovering your cells in LB instead of SOC?

I suspect your problem is in the SAP treatment, but it could also be the kinase reaction.  Try SAP treatment for shorter periods (or omit it) and see what happens.

Are you certain that the PCR enzyme in your kit produces blunt ends?

[sou]

phage434, on 02 December 2011 - 01:30 PM, said:

When you transform 1 ul of your 150 pg/ul DNA, how many colonies do you get?  Why are you recovering your cells in LB instead of SOC?

I suspect your problem is in the SAP treatment, but it could also be the kinase reaction.  Try SAP treatment for shorter periods (or omit it) and see what happens.

Are you certain that the PCR enzyme in your kit produces blunt ends?

when I tranasform 150pg DNA, the colonies I get are many (maybe 100-150) but not a lawn as I expected. so there is something wrong with my competent cells. (thx! i did not think of that till now! ) and what is wrong with recovering the cells in LB. I have done recovery in SOC before and I dont see any difference between the 2.
what can be the problem with the SAP treatment? (I thought its supposed to help me omit the background. thats good for me right? )
I think my SAP treatment is working fine 'coz there are no colonies on the control plate (vector, no insert ligation) I suspect its the kinase which is not working and hence no colonies on insert+vector plate. Am I thinking right here?
About the PCR... well i dont know if its giving me perfect blunt ends. its an RT-PCR kit from invitrogen with reverse transcriptase and taq polymerase. Taq does not give blunt ends?

[phage434]

For decent efficiency of 10^8 cfu/ug you should be getting about 15000 colonies, so your efficiency is quite low.  This alone could be your problem.  LB will work for recovery, but it's not standard, and you're looking to make things work as well and as standardly as possible.  SAP can not only dephosphorylate, but also trash the ends of DNA, so over digestion will give problems.  Less is more.  I'd be more concerned about failing to get any colonies in a ligation than in getting zero in the control.  We don't know if the kinase is working.  Taq does not typically give blunt ends, and could be another cause of your problems.