one end blunt end and other end sticky end-- ligation - vector with one end sticky and other blunt end (Aug/29/2005 )

hi all

i am trying to clone a gene from one virus to TMV -30B -Hisc vector
to get the histidine tag intact i can only use two restirction sites in the vector for cloning

but the problem is one end is blunt cutter and other is sticky
so ligation is not taking place properly


please help ??? any suggestions??


the sticky end is 3' over hang
can T4 pol merase help to fill in?? promega leaflet of the product claims so !!


or should i use klenow??


does inactivating the T4 pol require only 0.5M EDTA or should i heat inactivate too??


please help

laxmi

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I am having trouble understanding what you want to do. Can you explain the problem in more detail?

Daniel

Molecular biology protocols

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hello sir,


i am trying to clone the helper component gene of TEV to a vector named TMV-30B -HisC .
it has the movement protein and replicase of TMV but also a bit of TMGMV at the 3'end
i has only three restriction enzymes in the MCS

PacI PmeI and XhoI

i can only use PacI and PmeI since there is a Histine tag placed just outside pmeI site
and i need that tag with my protein

pacI is sticky and pme I is blunt end cutter

i tried double digestion , the enzymes are ordered from NEW ENGLAND BIOLAB , they have maximum efficiency in NEB Buffer 4

elution was done using a kit Nucleogen

but when i tried ligation it didnt work

so i thought maybe this could be the problem , since one end is sticky and other is blunt !!!

the original paper on the vector suggested filling in with T4 polymerase
i have promega 's T4 polymerase

only i am a bit confused about does T4 polymerase have filling up reaction from 3' to 5' direction
the promega leaflet says it can

was wondering ???

hope this was a bit clearer

i am attaching the paper about TMV vector
thanks

regards
laxmi

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Hello

I have a concern about DNA ligation.

I am trying to ligate 2 DNA fragments cut with kpn1 HindIII (1.3kb)and the other with KpnI and XhoI (6.3kb). I am only getting intramolecular ligations(2.6kb and 12.6kb and some other non specific bands).

I need to insert the ligated 7.4kb DNA in a vector cut with Hind III and XhoI and then clone it in E coli. I am using T4 DNA Ligase. Also its very difficult to detect the DNA in the gel as the quantity is too loo. What should I do about it.

Any suggestion will really be appreciated.

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Things are little clearer now but I do have a few more questions

What are the RE sites on your insert? Are they PacI and PmeI?
Did you gel purify your vector?
How did you generate your insert?

Daniel

DNA sequencing analysis software

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the restriction sites on the insert are PacI and Pme I .
i designed the primers for PCR of the gene with the restriction sites

PCR product was the required size when viewed thru agarose gel.
after restriction digestion , the products were extracted from the gel using kit .


i tried T4 polymerase filling in for both vector and insert ( promega) the concentrations were 1 microgram of DNA , according to the promega protocol

after that ligation using T4 ligase , over night at Room temperature
aslo used PEG in the reaction mixture
i transformed XL1-blue E.coli cells using heat shock method . the competent cells were made by CaCl2 method .
still only one or two colonies

some papers said that TMV vector is very unstable and difficult to get transformed colonies ,,


some suggested using klenow along with T4 pol
but i am not sure of the use

regards
laxmi

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I suggest that you clone your PCR insert into a PCR cloning vector like Topo then cut out the insert and clone it into your TMV vector. While this seems more complicated, in the long run it will be much simpler and less frustrating

I would also advise treating your RE digested TMV vector with shrimp alkaline phosphatase to prevent re-circularisation.

Daniel

LongTrace DNA sequencing software

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thank you for the suggestion sir

iw il try cloning into a new vector then

i already tried dephosphorylating TMV vector with calf intestine AP

thank you again

laxmi

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If I need to clone a PCR product into an expression vector this is the way I always do it.

The reason for using shrimp alkaline phosphatase is that it can be heat inactivated while calf AP is very heat stable. This makes it very difficult to remove from your reaction and it can carry over into the ligation step and dephosphorylate your insert.

Daniel

DNA sequencing protocols

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i see, i will try that too

i had one doubt , if T4 polymearse has 5' -> 3' filling in property will it work on 3' over hangs too??

or should i use exonuclease activity of T4 pol to make blunt end ?

thank you for ur valuable suggestions
regards
laxmi

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