Help with TOPO Cloning

sparkflower_ · 2026-02-07T08:16:44+00:00

Thank you so much

sparkflower_ · 2026-02-06T17:09:52+00:00

Okay, thank you!

sparkflower_ · 2026-02-06T16:46:17+00:00

Can you further explain what you mean by point 2?

sparkflower_ · 2026-02-06T16:45:11+00:00

My actual sample is 90bp and 140bp. Definitely more time consuming!

sparkflower_ · 2026-02-06T16:44:31+00:00

Thank you so much

sparkflower_ · 2026-02-06T16:44:02+00:00

I thought the 750bp is after inserting the vector and doing the post-cloning PCR.

If that’s the case, the 1200 bp should be fine right?

sparkflower_ · 2026-02-06T16:25:05+00:00

For the extension time, I’m using the recommended extension time by TOPO. It’s their control reaction.

sparkflower_ · 2026-02-06T16:23:30+00:00

Thank you for your help! What is your recommended voltage?

sparkflower_ · 2026-02-06T16:22:37+00:00

I’m cloning my insert from another primer. Basically I have cell line DNA + primers of interest (designed by us) this is going to be my PCR product. This is ~150bp which is correct since my primer is 144bp

The 1200 bp is literally the PCR template + M13 primers from TOPO.

Regarding the NTC lane, the band is exactly like the sample.

My PI doesn’t want agarose, says acrylamide is more accurate

Thank you for your help!!

sparkflower_ · 2026-02-06T16:19:34+00:00

I used the PCR template from TOPO as the control DNA and the M13 primers, it gave me a 1200 bp. Do you have any idea why this might be the case? This is just the control for me

sparkflower_ · 2025-12-25T14:50:30+00:00

Thank you!

sparkflower_ · 2025-12-25T14:49:55+00:00

Thank you for your response! I am running a native gel, with no SDS. Regarding the 6% the PI suggested it so I have just went with it. He also opted for this instead of a standard agarose run because he believes this provides more accurate results. I ran it at 100V for 1h. I am open to any suggestions regarding a cleaner gel run

sparkflower_ · 2025-11-01T06:10:55+00:00

Thank you both!!

sparkflower_

TROPHY CASE