I am generating bacmid in DH10 Bac cells. When I transformed the DH10 Bac cells, I got blue white colonies after 72 hr, but the colonies were very few just 4-5 white colonies. White colonies were restreaked and confirmed. In case of bacmid characterization by M13-For and rev PCR I get multiple bands along with the desired one. And upon transfecting the SF-9 cells and post pilot protein purification batch, I am getting multiple bands in his -tagged protein prep.
So is this problem is because of poor quality of bacmid ? How can I achieve the single specific band in PCR and ultimately better protein quality.
First try to perform some colony PCR to check the specificity.
Repeat the procedure of transfection to ensure proper transfection.
Try Gradient PCR to optimise the annealing and you can always try touchdown PCR program to ensure specificity as well
Given the multiple bands...looks like you need to optimize the PCR condition for the fragment. How big is your fragment ? Given only 4-5 white colonies something is not right here..you should be able to get plenty to play with. Another thing is what is the condition of DH10 Bac cells- were they the fresh thawed one or repeatedly freeze and thawed ?
Are you following Bac-to-Bac Topo expression system from invitrogen ?...It worked for me when I followed the exact protocol. Should work for you as well.
Here is the link for it: https://tools.lifetechnologies.com/content/sfs/manuals/bactobac_topo_exp_system_man.pdf
Hi there,
I agree with the above suggestions. Do not mix two possible problems (production of Bacmid and virus) and protein expression and purification together. Multiple bands with M13 Forward primer is a frequent problem but almost always can be resolved by playing with PCR conditions and the sequence of the target-specific (reverse) primer.
And, actually, I do not think that the recombination is a problem, unless you are trying to clone in a really big ORF (~3,000 bp). As to the purification on Ni-agarose, it may very often produce either partially purified material (hence, extra bands that belongs to untagged proteins) or partially degraded material (depending on the number of unstructured regions in your protein of interest and conditions of purification). I must tell here, that in my experience not all Sf-9 produced proteins can be easily purified under native conditions using 6His tag. In some cases I had to switch to Flag or HA tagged versions of my protein and obtained really pure and intact material though with a significantly lower overall yield. Try to include one more step of purification (size exclusion or ionic chromatography) if you have enough material upon elution from Ni. You can also try and do a small purification under denaturating conditions (6M GuCl) and see how clean your protein looks after that. If the protein is clean and more or less intact (>75-80%), then there is no problem with the corresponding Bacmid and you need to optimize purification conditions.
Best
For doing the PCR, it is recommended to use M13Forward OR Reverse and Gene of interest Forward OR Reverse.
M13 primers are short and there is a possibility of non-specific annealling.
By doing this, you will ensure that you have a bacmid with insertion of your gene of interest.
Hope this help.
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