Troubles with restriction digest of 5000 bp PCR product

[newmanreb]

Hi there, I'm having trouble with a restriction digest of a 5 kbp PCR product. The product was purified using a Qiagen PCR product clean up spin column kit. This product is too big to travel much on the gel, so I confirmed that it is the sequence I think it is by performing a nested PCR on the 5 kbp product to amplify a 300 bp product. This was successful. I am using a double digest of brand new NEB enzymes MseI and MluCI. These enzymes are TimeSaver certified (should digest in 5-15 minutes at 37 degrees) and both have 100% efficiency in the rCutSmart buffer. I set up half reactions of what NEB suggests with 0.50 ug DNA. I also tried doubling the amount of enzymes and doubling the time from 15 mins to 30 mins. Still, nothing I've tried results in the band on the gel changing. The main band remains relatively unchanged and there is a faint smear on the rest of the lane. I am stumped. I'm concerned that maybe the digest is working but I simply can't see any bands because these enzymes cut so many times? They should generate 49 fragments and the largest one is around 500 bp. I tried cutting this product with BamHI though as well and it didn't change anything on the gel even though it should generate fragments of 3456, 1088, and 364 for this sequence. Any advice on how to troubleshoot this would be greatly appreciated. Thanks in advance! 

Edited January 24, 2022 by newmanreb

[newmanreb]

Still confused about this if anyone is able to help me out! Thanks. 

[CharonY]

On 1/24/2022 at 3:23 PM, newmanreb said:

Hi there, I'm having trouble with a restriction digest of a 5 kbp PCR product. The product was purified using a Qiagen PCR product clean up spin column kit. This product is too big to travel much on the gel, so I confirmed that it is the sequence I think it is by performing a nested PCR on the 5 kbp product to amplify a 300 bp product. This was successful. I am using a double digest of brand new NEB enzymes MseI and MluCI. These enzymes are TimeSaver certified (should digest in 5-15 minutes at 37 degrees) and both have 100% efficiency in the rCutSmart buffer. I set up half reactions of what NEB suggests with 0.50 ug DNA. I also tried doubling the amount of enzymes and doubling the time from 15 mins to 30 mins. Still, nothing I've tried results in the band on the gel changing. The main band remains relatively unchanged and there is a faint smear on the rest of the lane. I am stumped. I'm concerned that maybe the digest is working but I simply can't see any bands because these enzymes cut so many times? They should generate 49 fragments and the largest one is around 500 bp. I tried cutting this product with BamHI though as well and it didn't change anything on the gel even though it should generate fragments of 3456, 1088, and 364 for this sequence. Any advice on how to troubleshoot this would be greatly appreciated. Thanks in advance! 

Did you use an appropriate gel percentage to see your fragments? Did the BamHI digest also show a smear? Was the undigested product smear-free? Was your estimate of the amount of DNA accurate? You could try to make prolonged digests (assuming no star activity) just to make sure. Finally if it is important, it might be worthwhile to submit your product for Sanger sequencing. Prices are so low now, it tends to be cheaper than the work time and material required to do all the trouble shooting and it gives at minimum confirmation that the ends are correct.