1916-pEC2-M-SssI

Cloning of 1916-pEC2-M-SssI

Purpose: Cloning of 1916-pEC2-M-SssI

Date:  2015-March 

Clone ID: 2412

Experimenters: Xu Xin

Results

cloning

Date: March 10,2015

use the primers 1916-M-SssI-1-1 and 1-6 to do a PCR I; 

use the primers 1916-M-SssI-1-7 and 2-14 to do a PCRII; 

 

Combined two PCR together

Then digest PCR product with NdeI and XhoI. Then clean up the digestion product. and ligation. transformation. selection 10 colonies to screen (PCR). 5 Colonies PCR result is great, take the five colonies to innoculate and extact plamid and Digestion. 

 

No product was found. Could be caused by the enzyme not working, use the home-made NdeI to test again.

Actually, none digestion could be caused by adding wrong amout buffer.

Fix Sequencing 3-20-15

Sent the sequencing, the result have the clone. But there is other mistake happening on oligo 2-13.

do the PCR 1-1 to 2-12,

Then purified the PCR puduct, use oligos 1-1, 2-12, and oligos 2-13 (1uM).

Digest the PCR product and vector 2015 PEC2 by NdeI/XhoI. purify the digested vector from Gel, also purify the PCR insert.

ligation, transformation. Screening:

  No.5 is the positive colony. Send to seqence 5-2-2015, and there is a mistake on oligos.

 1916-M-SssI-1-12/1-13.

Rescreening other 8 colonies:

1. 3 is the positive colony. Sent it to sequence on May 7 2015.

Refix the the sequencing #5 (sequenced on May 2, 2015)

There was a mistakes on oligos 1916-M-SssI-1-12/ 1-13

PCR fragment one: 1916-M-SssI-1-1 and 1916-M-SssI-1-12

PCR fragment two: 1916-M-SssI-1-13 and 1916-M-SssI-2-14

 Then purifiy two PCR fragments, and join them together.

Conducted a whole PCR using primers: 1916-M-SssI-1-1; 1916-M-SssI-2-14

The expected PCR product is 1200bp, but other PCR product (800bp) is very strong.

Reduce DNA template to 0.3ul in 25 PCR reaction, redo a PCR.

The band of 1200bp PCR is more strong than last one, so purified it from gel. digestion, ligation.

Screening:

_PCR.JPG

Sent #5 to sequence, the result wasn't good. there was a gap in the insert.Maybe the PCR step was't good.

Go back PCR, redo the clone.