2421-pEC-Thaumatin

Cloning of 2421-pEC-Thaumatin

Date:  2015-4

By: Xin

Results:

Gene Synthesis: about 750bp. The vector PEC (1454)

  Digested by NdeI /KpnI, ligation, transformation.

Screening:

ini-preparation

Send #2 to sequence on April 23,2015

Send #3 to sequence on April 25, 2015

Fix #3 on April 26, 2015:

use #3 sequenced on (April 25, 2015) as template,

PCR fragment I: the oligos 2421-Thaumatin-1 and 2421-Thaumatin-4

PCR fragment II: the oligos 2421-Thaumatin-5 and 2421-Thaumatin-18

Join two fragments together.

Screening:

 1,2,3 and 4 colonies are positive.

Send #4 to sequence on 5/5/2015, the sequencing is still same as the template.

Fix 5/5/2015

The template used#2  (sequenced on April 23,2015)

The mistakes was happened on oligo 2421-Thaumatin-1 / -2 

There are two ways to fix:

One is to use 2421-Thaumatin-1 and -18 redo a PCR.

The second way to fix is to do a PCR use 2421-Thaumatin-3 and -18.

Then use the PCR template to add 1umol 2421-Thaumatin-2 and use the primer is 2421-Thaumatin-1 and -18 do a big PCR.

Screening:

screening 4 colonies first (from Fix method one plate)

Send #4 to sequence, the result is still same as previous.

 Rescreening 8 colonies, 1-4 are the colonies conducted by fix method one; 5-8 are the colonies conducted by fix method two.

Send sequencing #6 to sequencing. There is still one mistake, which is different as previous mistakes. 

Rescreen 7 colonies (from fix method II plate).

 

Send #2 for sequencing on 5/15/2015.

Fix on 5/28/2015

Use the sequencing on 5-15-2015 as template:

The mistakes is on 2421-Thaumatin-4, so there are two PCR needed to fix the mistake.

The first one use primers 2421-Thaumatin-1 & 4

The second one use primers 2421-Thaumatin-5 & 18