Mysterious gain in vector size after cloning! - (Jul/06/2005 )

Hi everyone,

I'm having extremely strange results for my cloning reactions.

1. Foremost, I double digest my pENTR vector (2.7Kb) and pTOPO-insert (4.1Kb) with SalI (from NEB) and NotI (Promega).

2. Then I treat the digested vector with alkaline phosphatase.

3. I run gel, and got my linearised pENTR with overhang (2.3Kb) and my overhang insert (0.6Kb).

4. I excise the bands and extracted the DNA using Qiagen kit.

5. Then I ligate the 0.6Kb insert with pENTR (2.3Kb). I used 2uL for transformation, and the rest used to run gel. My gel result showed two bands, which are 2.3Kb (should be pENTR) and ~1.2Kb (insert dimers?? >> is this possible??)

6. Colonies grew on Kanamycin plate, and so I selected some colonies to scale up to 2mL and extract the plasmids using Miniprep.

7. Did a double digestion with SalI and NotI, run gel, and to my SUPRISE: 2 bands were seen (as expected) but the size were 0.6kb and ~3.5Kb. Well.. the 0.6Kb corresponds to the insert size, but my vector band should be 2.3Kb! How can it increase suddenly to 3.5Kb???

Anyone has any comments/ideas or soulation to my problem? Your help will be very much appreciated! Thank you so much!

Not sure exactly what is going on, but several things to consider.
1: Sal one is a poor cutter and doesn't cut supercoiled DNA well. Therefore, it is best to predigest with an enzyme then use it. Or, if possible, don't use it at all.
2: Yes, it is possible to get several inserts in tandem.
3: MAybe your digestion when checking your plasmid isn't complete.
4: Always sequence your final vector product.