2412 pEC2-AaeMutS

Cloning of 2412 pEC2-AaeMutS

Purpose: Cloning of 2412 pEC2-AaeMutS

'Date: 2015 March, April  

Clone ID: 2412

Experimenters:Xu Xin

cloning

Fix: 2412 sequecing: use the below primers to do 7 PCR, 

1-1 + 1-12; 1-13 + 1-16; 1-17 + 1-18; 1-19 + 2-14; 2-15 + 3-2 ; 3-3 + 3-6; 3-7 + 3-22

then 1-1 + 3-22

Cut gel for the 7 fragments, put together for gel purification. 

Use Olig for 1-1 and 3-22 for a big fragment PCR (annealing tem: 58, with should be 2500 bp, use 2x primer master mix).

 The PCR product was purified from gel, and digestion with vector 1454 by Nde1 and XhoI; ligation, transformation and screening.

The PCR screening only use 25 cycles, the band is hard to see.

But on the gel (in the dark), the colony 7, 8, 9 show a band clearly.

incolulation 7, 8, 9 and mini plasmid prepation, digested by XhoI + NdeI.

digested again with orginal 2412 plasmid to compare with

Repeat again on April 18, 2015,

Screening:

Digest by NdeI/XhoI and didn't find the expected cuting size, which could be caused the cutting size is too big (2500 bp).

Redigested by BamHI and NdeI, with 2412 positive control.The clonies# 2 and 6 have the expected product. 

 

The next step is sequencing.

Fixon April 28, 2015

The clony #6 was sequencd, there were still a few mistakes, and will be fixed.

Fragments	PCR Primers	Size
1	2412-AaeMutS-1-1; 1-14	600bp
2	2412-AaeMutS-1-17; 2-20	1021bp
3	2412-AaeMutS-1-1; 1-14	820bp

Join the three fragments together, using the purified PCR product,

10umol 2412-AaeMutS-1-1,

10umol 2412-AaeMutS-1-14,

1umol 2412-AaeMutS-3-1,

1umol 2412-AaeMutS-3-2,

1umol 2412-AaeMutS-3-3,

1umol 2412-AaeMutS-3-22.

2500bp PCR product was what we interested. Gel purified, digestion, ligation (5/7/2015)

Screening:

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Screening

1. 9 colony is the positive control

The colonies# 4,5,6 and 8 are positive. #4 was sent to sequencing on 5/10/15.

PCR again:

During experiment, the concentration (ng/ul) for oligos on plate 2-9-2015 were much lower than other oligo.

More oligos (on the plate 2-9-2015) were added into the PCR reaction, and carried PCR again.

During experiment, the concentration (ng/ul) for oligos on plate 2-9-2015 were much lower than other oligo.

More oligos (on the plate 2-9-2015) were added into the PCR reaction, and carried PCR again.

digestion, ligation, transformation.

Screening

3 is the positive colony. But this one didn't send to sequencing until the the sequencing result#4 come back and too many mistakes.

Fix on 5/18/2015

Use the #4 sequenced on 5/10/2015 as template to fix again.

There are three sections PCR was conducted:

1-1	1-16	700bp
1-17	3-2	1000bp
3-3	3-22	780bp

Combined all the PCR together as a template to carried a big PCR.