Excellgen at 21:45, 18 May 2015

2015-05-18T21:45:19Z

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== '''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; 

 [[File:1916-FIX-1ST&2ND-PCR-3-10-2015.JPG|151x254px|RTENOTITLE]]

Combined two PCR together

[[File:1916 2015 3 10 fix combined 1st and 2nd PCR.JPG|201x150px|RTENOTITLE]]

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. 

[[File:1916-fix-screening digestion-3-13-2015.JPG|142x142px|RTENOTITLE]] 

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.

<span style="font-size:larger">'''Fix Sequencing 3-20-15'''</span>

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).

[[File:2407 screening 4-1.JPG|203x210px|RTENOTITLE]]

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

[[File:Vector digested 4-29-15.JPG|RTENOTITLE]]

ligation, transformation. Screening:

'''''[[File:1916 2423 2421 screening.JPG|351x112px|RTENOTITLE]]'''''

  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:

[[File:1916.JPG|188x158px|RTENOTITLE]]

#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

[[File:2421;1916.JPG|RTENOTITLE]]

 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

[[File:1916,2421.JPG|181x130px|RTENOTITLE]]

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.

[[File:2412;21916.JPG|311x145px|RTENOTITLE]]

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

== '''''Screening''''': ==

[[File:1916-5.JPG|RTENOTITLE]]_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.

[[File:1916_PCR.JPG]]

1916-pEC2-M-SssI - Revision history

Excellgen at 21:45, 18 May 2015