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Cloning using BamH1 and HindIII - (Mar/14/2010 )

I m facing problem with BamH1 and HindIII double digestion.
As my primers dont have the overhang for the digetion with BamH1 and HindIII (restriction in my primers), I subclonned it thru TA cloning (PGEM-T easy vector Promega) so that I am sure about digestion.
As the BamH1 and HindIII suppled by NEB are not suitable for double digetion despite that I have very good band (300bp my Insert) after Double digestion in NEB2 buffer.
but for expression i want to clone it into pET28a(+).
But I am unable to have colonies after ligation (I have already check my ligation with controls).
One probability which strikes me is whether pET is being digested with both enzymes or not but if it is digetsed withj only one enzyme then at least I should have colonies after ligation due to self ligation.
Is it a problem of competent cells?
Help me.

-anilkumar-

What transfection method are you using with the pET28 vector?

-lab rat-

lab rat on Mar 15 2010, 06:05 AM said:

What transfection method are you using with the pET28 vector?

heat shock

-anilkumar-

Are you transforming into a cloning strain such as DH5a, or are you trying to directly transform BL21? The transformation efficiency of the BL21 strains is very low. In any case, your next experiment should be to quantify the transformation efficiency of your cells. They should be at least in the 10^8 cfu/ug region. Transform with 1 ul of serially diluted pUC19 at 10 pg/ul and get many colonies.

-phage434-

I don't see why HindIII-BamHI digestion should be any problem because that used to be a killer combo back during my PhD. Have you tested single digestion efficiency for both enzymes in all four NEB buffers? If you linearize your empty vector with HindIII or BamHI in four different reactions and buffer 1-4 (8 reactions in total plus control without enzyme)you can easily check which buffers that works and don't.

to me is sounds like something with the transfection efficiency. Have you checked your vector to insert ratios? Run a couple of Ál on a gel after ligation to check vector size. If your new vector has the correct size it got to be a transfection issue. Competent cells.....?

-DocFlow-

phage434 on Mar 22 2010, 05:17 AM said:

Are you transforming into a cloning strain such as DH5a, or are you trying to directly transform BL21? The transformation efficiency of the BL21 strains is very low. In any case, your next experiment should be to quantify the transformation efficiency of your cells. They should be at least in the 10^8 cfu/ug region. Transform with 1 ul of serially diluted pUC19 at 10 pg/ul and get many colonies.


I am using DH5a competent cells.
Now I am planning to use commercially available comp cells.

-anilkumar-

DocFlow on Mar 22 2010, 06:43 AM said:

I don't see why HindIII-BamHI digestion should be any problem because that used to be a killer combo back during my PhD. Have you tested single digestion efficiency for both enzymes in all four NEB buffers? If you linearize your empty vector with HindIII or BamHI in four different reactions and buffer 1-4 (8 reactions in total plus control without enzyme)you can easily check which buffers that works and don't.

to me is sounds like something with the transfection efficiency. Have you checked your vector to insert ratios? Run a couple of Ál on a gel after ligation to check vector size. If your new vector has the correct size it got to be a transfection issue. Competent cells.....?

BamH1 and HindIII give me a beautiful band of my insert at 300bp from pGEMT-easy (TA cloning)in Neb2 buffer but I am not sure of pET28a that is why now I switch over to sequential digestion for pET28a.
Waiting for the results

-anilkumar-

anilkumar on Mar 23 2010, 09:47 AM said:

DocFlow on Mar 22 2010, 06:43 AM said:

I don't see why HindIII-BamHI digestion should be any problem because that used to be a killer combo back during my PhD. Have you tested single digestion efficiency for both enzymes in all four NEB buffers? If you linearize your empty vector with HindIII or BamHI in four different reactions and buffer 1-4 (8 reactions in total plus control without enzyme)you can easily check which buffers that works and don't.

to me is sounds like something with the transfection efficiency. Have you checked your vector to insert ratios? Run a couple of Ál on a gel after ligation to check vector size. If your new vector has the correct size it got to be a transfection issue. Competent cells.....?

BamH1 and HindIII give me a beautiful band of my insert at 300bp from pGEMT-easy (TA cloning)in Neb2 buffer but I am not sure of pET28a that is why now I switch over to sequential digestion for pET28a.
Waiting for the results



i always had to do sequential digestion for Bam/Hind..... :o neb2 doesnt work for me... :D

-DRN-

DRN on Mar 23 2010, 06:38 AM said:

anilkumar on Mar 23 2010, 09:47 AM said:

DocFlow on Mar 22 2010, 06:43 AM said:

I don't see why HindIII-BamHI digestion should be any problem because that used to be a killer combo back during my PhD. Have you tested single digestion efficiency for both enzymes in all four NEB buffers? If you linearize your empty vector with HindIII or BamHI in four different reactions and buffer 1-4 (8 reactions in total plus control without enzyme)you can easily check which buffers that works and don't.

to me is sounds like something with the transfection efficiency. Have you checked your vector to insert ratios? Run a couple of Ál on a gel after ligation to check vector size. If your new vector has the correct size it got to be a transfection issue. Competent cells.....?

BamH1 and HindIII give me a beautiful band of my insert at 300bp from pGEMT-easy (TA cloning)in Neb2 buffer but I am not sure of pET28a that is why now I switch over to sequential digestion for pET28a.
Waiting for the results



i always had to do sequential digestion for Bam/Hind..... :D neb2 doesnt work for me... :D

I do digestion in Neb2 buffer but this time i incubate for longer period 5-6hrs
then ligation 1:3 n 1:5 ratio
Finally I got my clone.
now I have to go for sequencing meanwhile I am doing digestion to check whether I have my gene or not in vector.
Thanx for your support
Have a nice Day

-anilkumar-