arunee

Hi everybody,

I have been trying to clone a plasmid for months and now still can't get it. here is what i did.

I cut out my gene of interest from pUC57 plasmid using BamHI and SalI. It's about 1002 bp and there was a nice band on the gel.
Then i gel purified it. My vector is pcDNA4/HisMax from Invitrogen. The size is 5.3 kb. I cut the vector with BamHI and XhoI, then purified by Qiaqiuck purification kit (Qiagen) Then, ligated using T4 ligase from Invitrogen.
Here's the ligation mixture, (Insert:vector 3:1)

5x T4 ligase buffer 4  ul
T4 ligase                1  ul
Insert                      5 ul (57 ng)
Vector                    3 ul (100 ng)
D.W.                       7 ul
total volume           20 ul

The ligation was incubated at 25 hr for 3 hr and immediately transformed into TOP10 chemically competent cells.
Briefly, I used 5 ul ligation for transformation. The cells were incubated on ice for 30 min and heated shock at 42 c for 30 sec.
Then, i added 200 ul SOC medium into the tube and incubated the tube at 37 C for 2 hours shaking at 220 rpm.
The cells were plated on LB plate containing 100 ug Ampicillin, freshly prepared, and incubated at 37 C overnight.
The next morning, there were only 12 round nice colonies on my plate. The positive control i used was the uncut vector, which had numerous colonies while the negative one was D.W. which had no colony growth.

I picked all 12 single colonies and inoculated into 3 ml LB broth containing 100 ug/ml Ampicillin, freshly prepared as well, incubated at 37 C shaking at 220 rpm overnight. The plasmids were extracted by mini prep from Invitrogen.

I analyzed my plasmid using EcoRI and XbaI. The expected band was ~500 bp. (my insert had 1 unique EcoRI restriction site in the middle of the sequence, and the vector had 1 unique XbaI site).

But...... the DNA fragment I got was ~700 bp!

I tried cutting my insert out using BamHI and XhoI, there was nothing but the plasmid at around 6 kbp.

besides, one weird thing is, the pUC57plasmid which had my insert in it (I ordered commercially), no matter how hard I tried, I couldn't get a band from PCR! but I can cut my insert out every time. I tried sequencing, the insert was perfectly there!

So, here is my question,
1. Any idea where the 700 bp band came from?
2. how to solve PCR problem with the pUC57 plasmid i had?

Im very new to this field so any helps from you will be appreciated!!

Thank you