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#1 Travica

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Posted 27 May 2009 - 01:26 AM

Hello!
I would appreciate any advice you might have regarding my cloning problem.
I think I'm dealing with transformation problems, cause all the previous steps have been controled as much as possible..

I'm trying to clone 1500bp insert into pcDNA5/FRT vector (5070bp).
I've digested both with AflII and XhoI in NEB buffer4 and gel purified.
Both RE have been checked and they cut just fine in 100% buffer.

Vector was dephosphorilated and I have not noticed any concatemers on the gel.
I have used 10ng of vector in 1:1 and 1:3 molar ratios with insert, in 20ul ligation reaction.
ligation was performed on RT for 40mins with T4ligase.
in one of the attempts ligation reaction was frosen a day before transformation, but in most cases transformation procedeed right after ligation.

I have tried transforming Dh5alpha and Xl1blue with standard protocol, using:
5ul of ligation reaction
kept bact in ice 7min
heatshocked on 42C for 40-60s
back in ice 1min
added SOC (no antib) 200ul
shake on 37C 225rpm
apply 125ul on the ampicillin plates
o/n 37 incubate

Usually I get few healthy looking colonies on both ratio plates and on the negative control plate. Of course, sequencing shows no insert presence..

I have tried reducing the amount or DNA used for transformation, but then I get no colonies whatsoever.
I don't know if bacteria are particularly unhappy with my plasmid?!
They should be supercompetent, at least the Dh5alpha I've been using...
I've had no problems with my previous cloning using the different vector in the same bacteria.
pleeeease heeelp!!!
tnx

#2 Dr Teeth

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Posted 27 May 2009 - 10:52 AM

"ligation was performed on RT for 40mins with T4ligase"

Only 40 min at RT? I usually do 3-4 hrs at room temp with good success or overnight at 16C.


"heatshocked on 42C for 40-60s"

I heatshock DH5a at 42C for 20s.

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley

#3 eldon

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Posted 27 May 2009 - 04:18 PM

Hello!
I would appreciate any advice you might have regarding my cloning problem.
I think I'm dealing with transformation problems, cause all the previous steps have been controled as much as possible..

I'm trying to clone 1500bp insert into pcDNA5/FRT vector (5070bp).
I've digested both with AflII and XhoI in NEB buffer4 and gel purified.
Both RE have been checked and they cut just fine in 100% buffer.

Vector was dephosphorilated and I have not noticed any concatemers on the gel.
I have used 10ng of vector in 1:1 and 1:3 molar ratios with insert, in 20ul ligation reaction.
ligation was performed on RT for 40mins with T4ligase.
in one of the attempts ligation reaction was frosen a day before transformation, but in most cases transformation procedeed right after ligation.

I have tried transforming Dh5alpha and Xl1blue with standard protocol, using:
5ul of ligation reaction
kept bact in ice 7min
heatshocked on 42C for 40-60s
back in ice 1min
added SOC (no antib) 200ul
shake on 37C 225rpm
apply 125ul on the ampicillin plates
o/n 37 incubate

Usually I get few healthy looking colonies on both ratio plates and on the negative control plate. Of course, sequencing shows no insert presence..

I have tried reducing the amount or DNA used for transformation, but then I get no colonies whatsoever.
I don't know if bacteria are particularly unhappy with my plasmid?!
They should be supercompetent, at least the Dh5alpha I've been using...
I've had no problems with my previous cloning using the different vector in the same bacteria.
pleeeease heeelp!!!
tnx


ALWAYS perform uncut vector transformation controls or you will not know the relative competency of your cells...which varies greatly among kits or if you make your own stock of competent cells.

if your cells transform well with uncut vector, try two things:

1. prior to adding the ligase to your ligation reaction, heat the mixture for 5 min. at 50C and then immediately jam your tubes into ice...this will denature any H-bonds that have formed between compatible sticky ends and the ice will limit annealing of compatible ends that could result in concatemers.

2. extend the ligation reaction to all day (~ 8hrs) or overnight at 12 - 16C...sticky ends and TA ligations are favoured at lower temperatures...blunt at room temp is favoured.

if that doesn't work..consider your insert. does it have inverted repeats, extensive regions of low complexity or trinucleotide repeats? E. coli tends to dislike all 3 types of DNA. i work with huntingtin, and cloning fragments with 80+ CAG repeats is a horrible experience. however, there are tricks that make the cloning possible:

1. after transforming your cells, the recovery period in SOC and plating of the cells is done at room temp. analyzing transformants afterwards by mini-prep also requires growth at room temp....colony PCR is faster

2. reduce the ampicillin by half. copy number will be reduced in your transformants and so will any toxic effects.

These 2 tricks allowed me to clone huntingtin genes from many species and up to 118 CAG repeats without error.

#4 Travica

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Posted 29 May 2009 - 12:37 AM

Hello!
I would appreciate any advice you might have regarding my cloning problem.
I think I'm dealing with transformation problems, cause all the previous steps have been controled as much as possible..

I'm trying to clone 1500bp insert into pcDNA5/FRT vector (5070bp).
I've digested both with AflII and XhoI in NEB buffer4 and gel purified.
Both RE have been checked and they cut just fine in 100% buffer.

Vector was dephosphorilated and I have not noticed any concatemers on the gel.
I have used 10ng of vector in 1:1 and 1:3 molar ratios with insert, in 20ul ligation reaction.
ligation was performed on RT for 40mins with T4ligase.
in one of the attempts ligation reaction was frosen a day before transformation, but in most cases transformation procedeed right after ligation.

I have tried transforming Dh5alpha and Xl1blue with standard protocol, using:
5ul of ligation reaction
kept bact in ice 7min
heatshocked on 42C for 40-60s
back in ice 1min
added SOC (no antib) 200ul
shake on 37C 225rpm
apply 125ul on the ampicillin plates
o/n 37 incubate

Usually I get few healthy looking colonies on both ratio plates and on the negative control plate. Of course, sequencing shows no insert presence..

I have tried reducing the amount or DNA used for transformation, but then I get no colonies whatsoever.
I don't know if bacteria are particularly unhappy with my plasmid?!
They should be supercompetent, at least the Dh5alpha I've been using...
I've had no problems with my previous cloning using the different vector in the same bacteria.
pleeeease heeelp!!!
tnx


ALWAYS perform uncut vector transformation controls or you will not know the relative competency of your cells...which varies greatly among kits or if you make your own stock of competent cells.

if your cells transform well with uncut vector, try two things:

1. prior to adding the ligase to your ligation reaction, heat the mixture for 5 min. at 50C and then immediately jam your tubes into ice...this will denature any H-bonds that have formed between compatible sticky ends and the ice will limit annealing of compatible ends that could result in concatemers.

2. extend the ligation reaction to all day (~ 8hrs) or overnight at 12 - 16C...sticky ends and TA ligations are favoured at lower temperatures...blunt at room temp is favoured.

if that doesn't work..consider your insert. does it have inverted repeats, extensive regions of low complexity or trinucleotide repeats? E. coli tends to dislike all 3 types of DNA. i work with huntingtin, and cloning fragments with 80+ CAG repeats is a horrible experience. however, there are tricks that make the cloning possible:

1. after transforming your cells, the recovery period in SOC and plating of the cells is done at room temp. analyzing transformants afterwards by mini-prep also requires growth at room temp....colony PCR is faster

2. reduce the ampicillin by half. copy number will be reduced in your transformants and so will any toxic effects.

These 2 tricks allowed me to clone huntingtin genes from many species and up to 118 CAG repeats without error.



thank u for all suggestions.
I have done transf controls with uncut vector and I would usually get a carpet.
Yesterday I 've tried changing my cells, I ve used Top10 this time and got empty plates again..
I'll try prolonging the ligation time. The invitrogen Protocol I'm using is the "rapid lig" that suggests only 5 mins of ligation.
I've prolonged it to 45mins hoping it would help..maybe it'll work if I leave it whole day.
I'll let u know.
Good luck with fighting dinamic mutations and Good Job with successful cloning!!!!
tnx
sandra

#5 Travica

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Posted 29 May 2009 - 12:40 AM

"ligation was performed on RT for 40mins with T4ligase"

Only 40 min at RT? I usually do 3-4 hrs at room temp with good success or overnight at 16C.


"heatshocked on 42C for 40-60s"

I heatshock DH5a at 42C for 20s.



tnx for helping!
I'll reduce the heatshock time today.
Ligation is done by "rapid protocol" so it is suppose to be effective in 5mins..I'll try leaving it whole day.
cloning sucks..
:/




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