Hi Nusa,

It may be that an abnormal PCR product like the Primer dimer was inserted.

If you want to insert a very short DNA sequence such as 27 bp, you do not need to amplify the DNA by PCR.

First, prepare two types of oligomers suitable for the SpeI and SacI cleavage planes.

Probably, your target insertion sequence is as follows.

5'-AAGTTCTGGAATATGTGATCAAAGTG-3'.

Therefore, the oligomers you need are as follows

F: 5'-CTAAGTCACTTTTGATCACATATTCCAGAACTTTGAGCT-3'

R: 5'-CAAAGTTCTGGAATATGTGATCAAAGTGA-3'

These should bind complementarily and constitute a suitable protruding end for SpeI and SacI. Please confirm this.

I have used the following method for insertion.

Preparations

10 x Oligo annealing buffer

100 mM Tris-HCl, pH 8.0

10 mM EDTA, pH 8.0

1 M NaCl

Phagemid cleaved with SpeI and SacI

Procedure

1. anneal Oligomer using a thermal cycler　

100uM oligo F 4.5uL

100uM oligo R 4.5uL

10xOligo anealing buffer 1uL

Heat the mixture in a thermal cycler at 95°C for 4 minutes.

After heating, remove to room temperature. Be careful not to burn yourself.

Allow to cool at room temperature for 10 minutes.

By this operation, the above oligomer will bind to form a duplex.

2. ligation

Phagemid/SpeI/SacI 10~100ng

Anealed oligomer 1 uL

Add the appropriate amount of ligation reagent and ligate. 3.

Using the above reaction solution, introduce Phagemid into the competent cells by a general method.

Best,

Thank you very much for your advice! :):)

As I understand your protocol, therefore, I do not need to perform full PCR, but use 94 degrees only to form a duplex.

During preparations I see that you have mentioned Tris-HCl, EDTA, NaCl. Where I use all these chemicals?

Kind regards, Nuša

Tris-HCl, EDTA and NaCl are components of 10xOligo anealing buffer.

Alternatively, you can substitute the 10xOligo anealing buffer with NEB2.1 or Cutsmart buffer, which are buffers provided by restriction enzyme manufacturers.

Best,

Thank you!