Ligation/Transformation Troubles - Lentiviral vector (Jul/30/2010 )
Hi Guys,
I'm having a devil of a time trying to ligate a 3kb insert into an 8.9kb lentiviral vector. The vector and insert both contain unique Xba1 and EcoR1 sites. Here's what I've done so far:
1) Maxiprepped the empty lentiviral vector using a dam deficient bacteria, and confirmed the product's integrity by restriction enzymes.
2) Digested maxiprepped DNA vector with Xba1, gel purified the product
3) Digested gel purified product with EcoR1, gel purified the product
4) To get my insert, double digested with Xba1/EcoR1 using another vector
This is where my troubles seem to start (but please tell me if you think different):
5) Ligated insert with vector using T4 ligase at 16 deg o/n
6) Transformed ligation into SE-DH5alpha bacteria
7) Grew colonies, miniprepped, and double digested with Xba1/EcoR1 to check for insertion into the vector.
For 5-7, I transformed the following:
1) Insert + Xba1/EcoR1 cut vector from dam deficient bacteria, +T4 ligase --> around 10 colonies grew --> 1/8 colony harvests grew --> smear on agarose gel from miniprepped DNA/digested DNA
2) Insert + Xba1/EcoR1 cut vector from dam+ bacteria (SE-DH5alpha), +t4 ligase --> around 6 colonies grew --> 1/4 colony harvests grew --> smear on agarose gel from miniprepped DNA/digested DNA
3) Uncut vector --> hundreds of colonies grew
4) Xba1 cut vector from dam deficient bacteria, +T4 ligase --> ~50 colonies grew
I am missing a control in this ligation where I have Xba1/EcoR1 cut vector without any T4 ligase. However, I think it's peculiar that only 2 out of a total of 12 colonies actually grew up in my LB/ampicillin broth from the ligation transformations, and that both just showed up as smears on the gel.
My thoughts are that I should try using different bacteria, or that my LB/ampicillin plates have lost the antibiotic activity.
Any help?
Thanks!
Just an update if anyone has any thoughts.
I was able to transform XL1-blue bacteria with my ligation mixture and get colonies. 8/12 colonies grew up in LB broth, and I miniprepped and performed a restriction digest with Xba1 and EcoR1. All I found was empty lentiviral vector, no insert.
So to summerize:
Trying to ligate a 2.9kb insert into a ~9kb vector; insert was from a doubly digested prep. Vector from a sequential Xba1, EcoR1 digestion with gel purification. Ligation reaction was done at 3:1 and 6:1 molar ratios and transformed into XL1-blue bacteria. No colonies contained ligated insert by miniprep and Xba1/EcoR1 single or double digestion.
Thank you for your help, it would be much appreciated as I am extremely frustrated.
RPJ on Sat Jul 31 00:15:48 2010 said:
Hi Guys,
I'm having a devil of a time trying to ligate a 3kb insert into an 8.9kb lentiviral vector. The vector and insert both contain unique Xba1 and EcoR1 sites. Here's what I've done so far:
1) Maxiprepped the empty lentiviral vector using a dam deficient bacteria, and confirmed the product's integrity by restriction enzymes.
2) Digested maxiprepped DNA vector with Xba1, gel purified the product
3) Digested gel purified product with EcoR1, gel purified the product
4) To get my insert, double digested with Xba1/EcoR1 using another vector
This is where my troubles seem to start (but please tell me if you think different):
5) Ligated insert with vector using T4 ligase at 16 deg o/n
6) Transformed ligation into SE-DH5alpha bacteria
7) Grew colonies, miniprepped, and double digested with Xba1/EcoR1 to check for insertion into the vector.
For 5-7, I transformed the following:
1) Insert + Xba1/EcoR1 cut vector from dam deficient bacteria, +T4 ligase --> around 10 colonies grew --> 1/8 colony harvests grew --> smear on agarose gel from miniprepped DNA/digested DNA
2) Insert + Xba1/EcoR1 cut vector from dam+ bacteria (SE-DH5alpha), +t4 ligase --> around 6 colonies grew --> 1/4 colony harvests grew --> smear on agarose gel from miniprepped DNA/digested DNA
3) Uncut vector --> hundreds of colonies grew
4) Xba1 cut vector from dam deficient bacteria, +T4 ligase --> ~50 colonies grew
I am missing a control in this ligation where I have Xba1/EcoR1 cut vector without any T4 ligase. However, I think it's peculiar that only 2 out of a total of 12 colonies actually grew up in my LB/ampicillin broth from the ligation transformations, and that both just showed up as smears on the gel.
My thoughts are that I should try using different bacteria, or that my LB/ampicillin plates have lost the antibiotic activity.
Any help?
Thanks!
Maybe you just forgot to tell, but did you also gel purify your insert after double digestion? And what do the gels look like? Do you see your expected fragments ( i. e. does the digestion work)?
ElHo on Wed Aug 4 08:57:48 2010 said:
Maybe you just forgot to tell, but did you also gel purify your insert after double digestion? And what do the gels look like? Do you see your expected fragments ( i. e. does the digestion work)?
Sorry, yes I did gel purify my insert after double digestion, and I got the expected fragment. Single digested insert plasmid with Xba and Eco also gave the expected linearized plasmid.
XbaI and EcoRI ...both can be a pain in the neck ...XbaI can be impaired by methylation (you have to check that using a programm like NebCutter), EcoRI very often exerts star activity (maybe this explains the smear).
You should perform the following controls to check if everything is okay:
1) Vector only
2) Insert only
3) Vector+Ligase
4) Uncut vector the check comptency of your cells
5) Vector + Ligase
6) Insert + Ligase
I use to load 5+6on an agarose gel to see if you get a multiple bands by self-ligation ...more than the dimer-band ...than you can assume that insert and vector are digested properly. If this is not the case ligation is not possible and you have to digest longer, purify your DNA better and so on.
I dont know what competent cells u are using ...but when you re-liagte your XbaI cut vector you should get more than 50 colonies (if you use 50-100 ng vector)!!!
If the cells are good ...you should get a confluent plate with that apporach!
Do you buy them or prepare them yourself?
Regards,
p
RPJ on Mon Aug 2 20:41:52 2010 said:
Just an update if anyone has any thoughts.
I was able to transform XL1-blue bacteria with my ligation mixture and get colonies. 8/12 colonies grew up in LB broth, and I miniprepped and performed a restriction digest with Xba1 and EcoR1. All I found was empty lentiviral vector, no insert.
So to summerize:
Trying to ligate a 2.9kb insert into a ~9kb vector; insert was from a doubly digested prep. Vector from a sequential Xba1, EcoR1 digestion with gel purification. Ligation reaction was done at 3:1 and 6:1 molar ratios and transformed into XL1-blue bacteria. No colonies contained ligated insert by miniprep and Xba1/EcoR1 single or double digestion.
Thank you for your help, it would be much appreciated as I am extremely frustrated.
RPJ on Sat Jul 31 00:15:48 2010 said:
Hi Guys,
I'm having a devil of a time trying to ligate a 3kb insert into an 8.9kb lentiviral vector. The vector and insert both contain unique Xba1 and EcoR1 sites. Here's what I've done so far:
1) Maxiprepped the empty lentiviral vector using a dam deficient bacteria, and confirmed the product's integrity by restriction enzymes.
2) Digested maxiprepped DNA vector with Xba1, gel purified the product
3) Digested gel purified product with EcoR1, gel purified the product
4) To get my insert, double digested with Xba1/EcoR1 using another vector
This is where my troubles seem to start (but please tell me if you think different):
5) Ligated insert with vector using T4 ligase at 16 deg o/n
6) Transformed ligation into SE-DH5alpha bacteria
7) Grew colonies, miniprepped, and double digested with Xba1/EcoR1 to check for insertion into the vector.
For 5-7, I transformed the following:
1) Insert + Xba1/EcoR1 cut vector from dam deficient bacteria, +T4 ligase --> around 10 colonies grew --> 1/8 colony harvests grew --> smear on agarose gel from miniprepped DNA/digested DNA
2) Insert + Xba1/EcoR1 cut vector from dam+ bacteria (SE-DH5alpha), +t4 ligase --> around 6 colonies grew --> 1/4 colony harvests grew --> smear on agarose gel from miniprepped DNA/digested DNA
3) Uncut vector --> hundreds of colonies grew
4) Xba1 cut vector from dam deficient bacteria, +T4 ligase --> ~50 colonies grew
I am missing a control in this ligation where I have Xba1/EcoR1 cut vector without any T4 ligase. However, I think it's peculiar that only 2 out of a total of 12 colonies actually grew up in my LB/ampicillin broth from the ligation transformations, and that both just showed up as smears on the gel.
My thoughts are that I should try using different bacteria, or that my LB/ampicillin plates have lost the antibiotic activity.
Any help?
Thanks!
pDNA,
I've done controls 1, 3-6 you listed. I use SE-DH5a competent cells from invitrogen mainly. Uncut vector gives a confluent plate, while as I mentioned Religated Xba1 cut vector gives around 50 colonies. So I think the competency of the cells is fine. I wonder if it is possible my gel extraction somehow contains an inhibitor that is stopping ligation? I elute in Buffer EB (10mM Tris.Cl). DNA usually makes up 10-15uL of my 20uL ligation reaction.
My insert is from INV110 bacteria which are methylase deficient, so cutting with XbaI has not been a problem.
pDNA on Wed Aug 4 18:51:22 2010 said:
XbaI and EcoRI ...both can be a pain in the neck ...XbaI can be impaired by methylation (you have to check that using a programm like NebCutter), EcoRI very often exerts star activity (maybe this explains the smear).
You should perform the following controls to check if everything is okay:
1) Vector only
2) Insert only
3) Vector+Ligase
4) Uncut vector the check comptency of your cells
5) Vector + Ligase
6) Insert + Ligase
I use to load 5+6on an agarose gel to see if you get a multiple bands by self-ligation ...more than the dimer-band ...than you can assume that insert and vector are digested properly. If this is not the case ligation is not possible and you have to digest longer, purify your DNA better and so on.
I dont know what competent cells u are using ...but when you re-liagte your XbaI cut vector you should get more than 50 colonies (if you use 50-100 ng vector)!!!
If the cells are good ...you should get a confluent plate with that apporach!
Do you buy them or prepare them yourself?
Regards,
p
RPJ on Mon Aug 2 20:41:52 2010 said:
Just an update if anyone has any thoughts.
I was able to transform XL1-blue bacteria with my ligation mixture and get colonies. 8/12 colonies grew up in LB broth, and I miniprepped and performed a restriction digest with Xba1 and EcoR1. All I found was empty lentiviral vector, no insert.
So to summerize:
Trying to ligate a 2.9kb insert into a ~9kb vector; insert was from a doubly digested prep. Vector from a sequential Xba1, EcoR1 digestion with gel purification. Ligation reaction was done at 3:1 and 6:1 molar ratios and transformed into XL1-blue bacteria. No colonies contained ligated insert by miniprep and Xba1/EcoR1 single or double digestion.
Thank you for your help, it would be much appreciated as I am extremely frustrated.
RPJ on Sat Jul 31 00:15:48 2010 said:
Hi Guys,
I'm having a devil of a time trying to ligate a 3kb insert into an 8.9kb lentiviral vector. The vector and insert both contain unique Xba1 and EcoR1 sites. Here's what I've done so far:
1) Maxiprepped the empty lentiviral vector using a dam deficient bacteria, and confirmed the product's integrity by restriction enzymes.
2) Digested maxiprepped DNA vector with Xba1, gel purified the product
3) Digested gel purified product with EcoR1, gel purified the product
4) To get my insert, double digested with Xba1/EcoR1 using another vector
This is where my troubles seem to start (but please tell me if you think different):
5) Ligated insert with vector using T4 ligase at 16 deg o/n
6) Transformed ligation into SE-DH5alpha bacteria
7) Grew colonies, miniprepped, and double digested with Xba1/EcoR1 to check for insertion into the vector.
For 5-7, I transformed the following:
1) Insert + Xba1/EcoR1 cut vector from dam deficient bacteria, +T4 ligase --> around 10 colonies grew --> 1/8 colony harvests grew --> smear on agarose gel from miniprepped DNA/digested DNA
2) Insert + Xba1/EcoR1 cut vector from dam+ bacteria (SE-DH5alpha), +t4 ligase --> around 6 colonies grew --> 1/4 colony harvests grew --> smear on agarose gel from miniprepped DNA/digested DNA
3) Uncut vector --> hundreds of colonies grew
4) Xba1 cut vector from dam deficient bacteria, +T4 ligase --> ~50 colonies grew
I am missing a control in this ligation where I have Xba1/EcoR1 cut vector without any T4 ligase. However, I think it's peculiar that only 2 out of a total of 12 colonies actually grew up in my LB/ampicillin broth from the ligation transformations, and that both just showed up as smears on the gel.
My thoughts are that I should try using different bacteria, or that my LB/ampicillin plates have lost the antibiotic activity.
Any help?
Thanks!
it is also possible that your overhangs have been wrecked during UV exposure during gel purification.
Regards,
p
RPJ on Wed Aug 4 19:14:56 2010 said:
pDNA,
I've done controls 1, 3-6 you listed. I use SE-DH5a competent cells from invitrogen mainly. Uncut vector gives a confluent plate, while as I mentioned Religated Xba1 cut vector gives around 50 colonies. So I think the competency of the cells is fine. I wonder if it is possible my gel extraction somehow contains an inhibitor that is stopping ligation? I elute in Buffer EB (10mM Tris.Cl). DNA usually makes up 10-15uL of my 20uL ligation reaction.
My insert is from INV110 bacteria which are methylase deficient, so cutting with XbaI has not been a problem.
pDNA on Wed Aug 4 18:51:22 2010 said:
XbaI and EcoRI ...both can be a pain in the neck ...XbaI can be impaired by methylation (you have to check that using a programm like NebCutter), EcoRI very often exerts star activity (maybe this explains the smear).
You should perform the following controls to check if everything is okay:
1) Vector only
2) Insert only
3) Vector+Ligase
4) Uncut vector the check comptency of your cells
5) Vector + Ligase
6) Insert + Ligase
I use to load 5+6on an agarose gel to see if you get a multiple bands by self-ligation ...more than the dimer-band ...than you can assume that insert and vector are digested properly. If this is not the case ligation is not possible and you have to digest longer, purify your DNA better and so on.
I dont know what competent cells u are using ...but when you re-liagte your XbaI cut vector you should get more than 50 colonies (if you use 50-100 ng vector)!!!
If the cells are good ...you should get a confluent plate with that apporach!
Do you buy them or prepare them yourself?
Regards,
p
RPJ on Mon Aug 2 20:41:52 2010 said:
Just an update if anyone has any thoughts.
I was able to transform XL1-blue bacteria with my ligation mixture and get colonies. 8/12 colonies grew up in LB broth, and I miniprepped and performed a restriction digest with Xba1 and EcoR1. All I found was empty lentiviral vector, no insert.
So to summerize:
Trying to ligate a 2.9kb insert into a ~9kb vector; insert was from a doubly digested prep. Vector from a sequential Xba1, EcoR1 digestion with gel purification. Ligation reaction was done at 3:1 and 6:1 molar ratios and transformed into XL1-blue bacteria. No colonies contained ligated insert by miniprep and Xba1/EcoR1 single or double digestion.
Thank you for your help, it would be much appreciated as I am extremely frustrated.
RPJ on Sat Jul 31 00:15:48 2010 said:
Hi Guys,
I'm having a devil of a time trying to ligate a 3kb insert into an 8.9kb lentiviral vector. The vector and insert both contain unique Xba1 and EcoR1 sites. Here's what I've done so far:
1) Maxiprepped the empty lentiviral vector using a dam deficient bacteria, and confirmed the product's integrity by restriction enzymes.
2) Digested maxiprepped DNA vector with Xba1, gel purified the product
3) Digested gel purified product with EcoR1, gel purified the product
4) To get my insert, double digested with Xba1/EcoR1 using another vector
This is where my troubles seem to start (but please tell me if you think different):
5) Ligated insert with vector using T4 ligase at 16 deg o/n
6) Transformed ligation into SE-DH5alpha bacteria
7) Grew colonies, miniprepped, and double digested with Xba1/EcoR1 to check for insertion into the vector.
For 5-7, I transformed the following:
1) Insert + Xba1/EcoR1 cut vector from dam deficient bacteria, +T4 ligase --> around 10 colonies grew --> 1/8 colony harvests grew --> smear on agarose gel from miniprepped DNA/digested DNA
2) Insert + Xba1/EcoR1 cut vector from dam+ bacteria (SE-DH5alpha), +t4 ligase --> around 6 colonies grew --> 1/4 colony harvests grew --> smear on agarose gel from miniprepped DNA/digested DNA
3) Uncut vector --> hundreds of colonies grew
4) Xba1 cut vector from dam deficient bacteria, +T4 ligase --> ~50 colonies grew
I am missing a control in this ligation where I have Xba1/EcoR1 cut vector without any T4 ligase. However, I think it's peculiar that only 2 out of a total of 12 colonies actually grew up in my LB/ampicillin broth from the ligation transformations, and that both just showed up as smears on the gel.
My thoughts are that I should try using different bacteria, or that my LB/ampicillin plates have lost the antibiotic activity.
Any help?
Thanks!
That was one of my thoughts also, so in the past couple days I did a ligation with newly cut insert and vector. I still get very few colonies on my insert+vector plate so I am not optimistic. But, I am running a gel right now to see if any insert is present...we'll see how this goes.
pDNA on Wed Aug 4 19:21:32 2010 said:
it is also possible that your overhangs have been wrecked during UV exposure during gel purification.
Regards,
p
RPJ on Wed Aug 4 19:14:56 2010 said:
pDNA,
I've done controls 1, 3-6 you listed. I use SE-DH5a competent cells from invitrogen mainly. Uncut vector gives a confluent plate, while as I mentioned Religated Xba1 cut vector gives around 50 colonies. So I think the competency of the cells is fine. I wonder if it is possible my gel extraction somehow contains an inhibitor that is stopping ligation? I elute in Buffer EB (10mM Tris.Cl). DNA usually makes up 10-15uL of my 20uL ligation reaction.
My insert is from INV110 bacteria which are methylase deficient, so cutting with XbaI has not been a problem.
pDNA on Wed Aug 4 18:51:22 2010 said:
XbaI and EcoRI ...both can be a pain in the neck ...XbaI can be impaired by methylation (you have to check that using a programm like NebCutter), EcoRI very often exerts star activity (maybe this explains the smear).
You should perform the following controls to check if everything is okay:
1) Vector only
2) Insert only
3) Vector+Ligase
4) Uncut vector the check comptency of your cells
5) Vector + Ligase
6) Insert + Ligase
I use to load 5+6on an agarose gel to see if you get a multiple bands by self-ligation ...more than the dimer-band ...than you can assume that insert and vector are digested properly. If this is not the case ligation is not possible and you have to digest longer, purify your DNA better and so on.
I dont know what competent cells u are using ...but when you re-liagte your XbaI cut vector you should get more than 50 colonies (if you use 50-100 ng vector)!!!
If the cells are good ...you should get a confluent plate with that apporach!
Do you buy them or prepare them yourself?
Regards,
p
RPJ on Mon Aug 2 20:41:52 2010 said:
Just an update if anyone has any thoughts.
I was able to transform XL1-blue bacteria with my ligation mixture and get colonies. 8/12 colonies grew up in LB broth, and I miniprepped and performed a restriction digest with Xba1 and EcoR1. All I found was empty lentiviral vector, no insert.
So to summerize:
Trying to ligate a 2.9kb insert into a ~9kb vector; insert was from a doubly digested prep. Vector from a sequential Xba1, EcoR1 digestion with gel purification. Ligation reaction was done at 3:1 and 6:1 molar ratios and transformed into XL1-blue bacteria. No colonies contained ligated insert by miniprep and Xba1/EcoR1 single or double digestion.
Thank you for your help, it would be much appreciated as I am extremely frustrated.
RPJ on Sat Jul 31 00:15:48 2010 said:
Hi Guys,
I'm having a devil of a time trying to ligate a 3kb insert into an 8.9kb lentiviral vector. The vector and insert both contain unique Xba1 and EcoR1 sites. Here's what I've done so far:
1) Maxiprepped the empty lentiviral vector using a dam deficient bacteria, and confirmed the product's integrity by restriction enzymes.
2) Digested maxiprepped DNA vector with Xba1, gel purified the product
3) Digested gel purified product with EcoR1, gel purified the product
4) To get my insert, double digested with Xba1/EcoR1 using another vector
This is where my troubles seem to start (but please tell me if you think different):
5) Ligated insert with vector using T4 ligase at 16 deg o/n
6) Transformed ligation into SE-DH5alpha bacteria
7) Grew colonies, miniprepped, and double digested with Xba1/EcoR1 to check for insertion into the vector.
For 5-7, I transformed the following:
1) Insert + Xba1/EcoR1 cut vector from dam deficient bacteria, +T4 ligase --> around 10 colonies grew --> 1/8 colony harvests grew --> smear on agarose gel from miniprepped DNA/digested DNA
2) Insert + Xba1/EcoR1 cut vector from dam+ bacteria (SE-DH5alpha), +t4 ligase --> around 6 colonies grew --> 1/4 colony harvests grew --> smear on agarose gel from miniprepped DNA/digested DNA
3) Uncut vector --> hundreds of colonies grew
4) Xba1 cut vector from dam deficient bacteria, +T4 ligase --> ~50 colonies grew
I am missing a control in this ligation where I have Xba1/EcoR1 cut vector without any T4 ligase. However, I think it's peculiar that only 2 out of a total of 12 colonies actually grew up in my LB/ampicillin broth from the ligation transformations, and that both just showed up as smears on the gel.
My thoughts are that I should try using different bacteria, or that my LB/ampicillin plates have lost the antibiotic activity.
Any help?
Thanks!
then Good luck!!!
Let us know if u have been successful!
Maybe you can use a unique restriction enzyme that cuts only in your insert to use for the control digest instead of the double digest with EcoRI and XbaI.
Regards,
p
RPJ on Wed Aug 4 20:07:09 2010 said:
That was one of my thoughts also, so in the past couple days I did a ligation with newly cut insert and vector. I still get very few colonies on my insert+vector plate so I am not optimistic. But, I am running a gel right now to see if any insert is present...we'll see how this goes.
pDNA on Wed Aug 4 19:21:32 2010 said:
it is also possible that your overhangs have been wrecked during UV exposure during gel purification.
Regards,
p
RPJ on Wed Aug 4 19:14:56 2010 said:
pDNA,
I've done controls 1, 3-6 you listed. I use SE-DH5a competent cells from invitrogen mainly. Uncut vector gives a confluent plate, while as I mentioned Religated Xba1 cut vector gives around 50 colonies. So I think the competency of the cells is fine. I wonder if it is possible my gel extraction somehow contains an inhibitor that is stopping ligation? I elute in Buffer EB (10mM Tris.Cl). DNA usually makes up 10-15uL of my 20uL ligation reaction.
My insert is from INV110 bacteria which are methylase deficient, so cutting with XbaI has not been a problem.
pDNA on Wed Aug 4 18:51:22 2010 said:
XbaI and EcoRI ...both can be a pain in the neck ...XbaI can be impaired by methylation (you have to check that using a programm like NebCutter), EcoRI very often exerts star activity (maybe this explains the smear).
You should perform the following controls to check if everything is okay:
1) Vector only
2) Insert only
3) Vector+Ligase
4) Uncut vector the check comptency of your cells
5) Vector + Ligase
6) Insert + Ligase
I use to load 5+6on an agarose gel to see if you get a multiple bands by self-ligation ...more than the dimer-band ...than you can assume that insert and vector are digested properly. If this is not the case ligation is not possible and you have to digest longer, purify your DNA better and so on.
I dont know what competent cells u are using ...but when you re-liagte your XbaI cut vector you should get more than 50 colonies (if you use 50-100 ng vector)!!!
If the cells are good ...you should get a confluent plate with that apporach!
Do you buy them or prepare them yourself?
Regards,
p
RPJ on Mon Aug 2 20:41:52 2010 said:
Just an update if anyone has any thoughts.
I was able to transform XL1-blue bacteria with my ligation mixture and get colonies. 8/12 colonies grew up in LB broth, and I miniprepped and performed a restriction digest with Xba1 and EcoR1. All I found was empty lentiviral vector, no insert.
So to summerize:
Trying to ligate a 2.9kb insert into a ~9kb vector; insert was from a doubly digested prep. Vector from a sequential Xba1, EcoR1 digestion with gel purification. Ligation reaction was done at 3:1 and 6:1 molar ratios and transformed into XL1-blue bacteria. No colonies contained ligated insert by miniprep and Xba1/EcoR1 single or double digestion.
Thank you for your help, it would be much appreciated as I am extremely frustrated.
RPJ on Sat Jul 31 00:15:48 2010 said:
Hi Guys,
I'm having a devil of a time trying to ligate a 3kb insert into an 8.9kb lentiviral vector. The vector and insert both contain unique Xba1 and EcoR1 sites. Here's what I've done so far:
1) Maxiprepped the empty lentiviral vector using a dam deficient bacteria, and confirmed the product's integrity by restriction enzymes.
2) Digested maxiprepped DNA vector with Xba1, gel purified the product
3) Digested gel purified product with EcoR1, gel purified the product
4) To get my insert, double digested with Xba1/EcoR1 using another vector
This is where my troubles seem to start (but please tell me if you think different):
5) Ligated insert with vector using T4 ligase at 16 deg o/n
6) Transformed ligation into SE-DH5alpha bacteria
7) Grew colonies, miniprepped, and double digested with Xba1/EcoR1 to check for insertion into the vector.
For 5-7, I transformed the following:
1) Insert + Xba1/EcoR1 cut vector from dam deficient bacteria, +T4 ligase --> around 10 colonies grew --> 1/8 colony harvests grew --> smear on agarose gel from miniprepped DNA/digested DNA
2) Insert + Xba1/EcoR1 cut vector from dam+ bacteria (SE-DH5alpha), +t4 ligase --> around 6 colonies grew --> 1/4 colony harvests grew --> smear on agarose gel from miniprepped DNA/digested DNA
3) Uncut vector --> hundreds of colonies grew
4) Xba1 cut vector from dam deficient bacteria, +T4 ligase --> ~50 colonies grew
I am missing a control in this ligation where I have Xba1/EcoR1 cut vector without any T4 ligase. However, I think it's peculiar that only 2 out of a total of 12 colonies actually grew up in my LB/ampicillin broth from the ligation transformations, and that both just showed up as smears on the gel.
My thoughts are that I should try using different bacteria, or that my LB/ampicillin plates have lost the antibiotic activity.
Any help?
Thanks!
pDNA on Wed Aug 4 20:17:23 2010 said:
then Good luck!!!
Let us know if u have been successful!
Maybe you can use a unique restriction enzyme that cuts only in your insert to use for the control digest instead of the double digest with EcoRI and XbaI.
Regards,
p
RPJ on Wed Aug 4 20:07:09 2010 said:
That was one of my thoughts also, so in the past couple days I did a ligation with newly cut insert and vector. I still get very few colonies on my insert+vector plate so I am not optimistic. But, I am running a gel right now to see if any insert is present...we'll see how this goes.
pDNA on Wed Aug 4 19:21:32 2010 said:
it is also possible that your overhangs have been wrecked during UV exposure during gel purification.
Regards,
p
Probable success! My plate had 4 colonies on it. 2 grew up in liquid culture. 1/2 plasmids had a gel shift when running against empty vector. So, I digested with EcoR1/Xba1. Thinking that Xba site may be methylated in the ligated product although it is unmethylated in the empty vector, I also digested with EcoR1/BamH1. I did not see the right fragment with Eco/Xba, so probable methylation now in the ligated product. But, I did see the right restriction products with Eco/Bam. The next step for me is to grow this plasmid up in a methylase deficient bacteria for the gold standard Xba/Eco test, and also some PCR for insert. Thanks for the help guys - lets hope there was no recombination occuring below the radar....