How to transform with topo vector - (Oct/18/2008 )
I have been trying champion pet101 topo vector (Invitrogen) for transformation in competent Top 10 e coli cells provided with kit, but it failed every time.
1) I think there is a problem with the competent cells, they might have lost this property,
Kindly suggest me if there is a better way to ligate topo vector and PCR product and transform it into E coli other than kit protocol
1) I think there is a problem with the competent cells, they might have lost this property,
Kindly suggest me if there is a better way to ligate topo vector and PCR product and transform it into E coli other than kit protocol
Don't think that there's a problem. . . Prove it.
Then we can kindly proceed to the other alternative.
1) I think there is a problem with the competent cells, they might have lost this property,
Kindly suggest me if there is a better way to ligate topo vector and PCR product and transform it into E coli other than kit protocol
Don't just think that there's a problem. . . Prove it. show controls ( - and +)
Then we can kindly proceed to the other alternative.
Actually my protocol involves,
setting up topo reaction (vector : dna ratio = 1:1) Vector only control
water 3ul water 4ul
pcr product 1ul (5ng) pcr product 0ul
salt solution 1ul salt solution 1ul
topo vector 1ul topo vector 1ul
mix reac. gently and incubate for 30 min at 22.5 C
transformation 1 shot top10 e coli cells
1)add 3ul topo reaction into a vial of 1 shot e coli cells
2) mix gently
3) incubate on ice for 30 min
4) heat shock for 30 sec at 42C
5) transfer tubes to ice
6)Add 250ul soc media
7)shake for 1 hr at 37C
8)spread 100-200ul & incubate overnight at 37C
I invite any suggestions which could help me out,
cheers
setting up topo reaction (vector : dna ratio = 1:1) Vector only control
water 3ul water 4ul
pcr product 1ul (5ng) pcr product 0ul
salt solution 1ul salt solution 1ul
topo vector 1ul topo vector 1ul
mix reac. gently and incubate for 30 min at 22.5 C
transformation 1 shot top10 e coli cells
1)add 3ul topo reaction into a vial of 1 shot e coli cells
2) mix gently
3) incubate on ice for 30 min
4) heat shock for 30 sec at 42C
5) transfer tubes to ice
6)Add 250ul soc media
7)shake for 1 hr at 37C
8)spread 100-200ul & incubate overnight at 37C
I invite any suggestions which could help me out,
cheers
you will need something other than the vector. topo vector is a linearized plasmid with topoisomerase protein attached the to ends. TOPO kit from invitrogen usually included a vector like pUC19 for control purpose to help you check for your transformation efficiency ...
thank you dear for ur valuable suggestions,
finally i have got the results with the old technique
finally i have got the results with the old technique
Hi Reuben , Iam also trying to clone a PCR product into a Blunt topo vector, however,I triedseveraltime andI do not get any results. All I got is a band, several, all the same size which does not correspond to my insert. I dont know what to do,could you help me????
Thank you