3 replies to this topic

[vinnie1221]

Dear All

I am trying to insert a 1kb fragment into the vector PUAST (~9kb). Unfortunately, I failed three times in a row but have no clue where did I go wrong. Please give me some suggestions if you have worked /are working on similar cases.

My protocol is the following:

I started with extracting my insert & vector via mini prep. The concetrations for both are roughly 300-400ng/ul

Then I did a digestion for both vector and insert ( vector- 1ug, insert - 5ug) with the restriction enzyme EcROI and NotI, Buffer 3, 2Hrs at 37oC. Heat Inactivate for 15 minutes at 80oC

After that I did a gel extraction for the insert and also gel purifiied it straight after.

For the ligation, I used 2ul vector, 1ul ligase, 4ul ligate buffer and 13ul insert, 15oC overnight

After the transformation, I did a screening by picking 40 colonies and none of them showed a positive result.

The second time, my boss suggested to use more amount of DNA for the insert, so I doubled it to 10ug and other conditions kept the same. However, negatiive results again

The third time, I did not do the gel extraction for the insert as I heard the gel extraction may affect the ligation process. Instead, I did a double digestion with ECoRI and NotI for the first digestion and NCOI for the second. Other conditions were unchanged. failed again.

I am trying very hard on this clone but really have no idea what should I do next. It will be great if somebody can help.
Thank you in advance.

Vincent

[vinnie1221]

the restriction enzyme should be EcoRI. Sorry about that

[il0postino]

Vincent,

So you work with flies!

thought to just give you a suggestion protocol:

start with  3 ug of DNA of vector plasmid and 4.5 ug of insert plasmid. Digest each with EcoRI and NotI using NEB buff3 in ~100-150 ul total volume (with a bit of volume you dilute out possible impurities in the mini prep) use 2.5 ul of each enzyme (~10x overdigetion)
After 1-1.5 hours add CIP (calf intestine alkaline phosphatase) to the vector tube only (~2units) mix well and incubate both tubes for  30-45 minutes longer (also at 37C). This will reduce the background (also when using 2 different enzymes). Add gel loading buffer and load the two samples directly on a gel using multiple slots for each sample -because of the volume and to prevent overloading of the gel (for 9 kb use a gel below 1% agarose). Cut out the vector and insert bands carefully (if it is still overloaded just take the front of the band, also, use a long wave length UV box eg 360nm -DNA irradiated with short wave length UV will not clone).
Isolate the DNA from the gel slices and resuspend/collect in a small volume.
Ligate using 1.5 ul of each (will be appr. 1:3 molar ratio) in a total volume of 10ul -using 1 ul of 10x lig buffer 5.25 h2o and 0.75 T4 ligase. ligate o/n at 16C. Also take along controls: one  using 1.5 h20 instead of vector and one  using 1.5 h2o instead of insert.
Transform the most competent bacteria you have (2.5 ul lig  in 50ul cells) and plate 2 dilutions of each transformation (to ensure single colonies on at least one plate).
Determine if the ligation shows enrichment as compared to the control plates.
If it looks good: Pick ~6 free colonies (pick colonies of all sizes present on the plate)
analyze and celebrate (positive thinking helps too!)

[vinnie1221]

Thank you very much. I defintely will try your protocol today. Hope will give you some good news soon