Hi Syafiqah Ishak

1) Most likely. I notice you have used 0.4x buffer in your 30uL reaction (6*2/30). This is very low and could have resulted in star activity. Your enzyme is also at the 5% glycerol threshold (3 uL in 30 uL), which means a high enzyme to volume ratio. Under this sub-optimal buffer conditions, the risk of star activity or non-specific cutting further increased. This explains the additional band you observed.

2. Very unlikely since both BamHI and EcoRI are usually single-site in the MCS for pcDNA3.1-HisC.

3. I usually follow this rule: for a 50 uL reaction, use 1 ug DNA and 1 unit of enzyme per 1 ug DNA, unless you need to scale down to save reagents. In your case, since you already used at least 1 uL of enzyme in the reaction, I don’t think scaling down applies.

Try this: 25 uL of 2x buffer, 1 uL each of BamHI and EcoRI, 1 ug DNA, and water up to 50 uL (in the same order you usually add components, just make sure the buffer is at 1x final concentration before adding the enzymes). Incubate for 2 hours at 37 °C. Do not heat-inactivate. I dont think it's necessary.

I would recommend you to include proper control to ensure you interpret the digest result properly. include undigested control and single digest control.

4. Do you plan to perform ligation afterwards? If so, I suggest not worrying too much about the extra ~750 bp band. It’s most likely due to star activity or an unspecific byproduct. Just excise and extract the bands corresponding to your target vector and insert sizes.

There are some good points made by Alif Ismail be sure to check your buffer, usually restriction buffers come as 10X stock and not 2X, but be sure you are using the right amount for your volume. Having the wrong buffer will change the salt concentration and can have an effect on accuracy of digestion.

Secondly, you are using a lot of enzyme. Most enzymes often come as 10-20 units per microliter, so you only need a fraction of a microliter. One microliter is a volume, not a unit measurement, you need to see how much enzyme you have per volume. So those 2 things can result in getting some digestion at secondary sites.

Lastly though, you have very little of your vector DNA on the gel, I would run the gel again with a LOT more of the vector. It might be that the 750 bp band is actually there and that is telling you that there might be a problem with your vector.