Editing 1151 pET30b-sso7d-ToughTaq (section)

== '''Results'''  ==

=== Cloning  ===

Date:&nbsp;June 13, 2012 

Synthesized template in 3 fragments (KTaq1, KTaq1 and KTaq3), PCR with KAPA&nbsp;enzyme for 5 cycles with 15 seconds extension time, conbined them, added first (KTaq1-1) and last (KTaq3-14) oligos , PCR with KAPA&nbsp;enzyme for 20 cycles with 45 seconds extension time. Ran gel (lanes 1-4), supposed to be ~1.7 kb 

[[Image:0613 Amplifacation.JPG]] 

Reamplified 1151 (see lanes 1-3) in fragments <br> 

Lane 1:&nbsp;KTaq1-1 - KTaq1-16 (636 bp)<br> 

Lane 2:&nbsp;KTaq2-1 - KTaq2-16 (629 bp)<br> 

Lane 3:&nbsp;KTaq3-1 - KTaq3-14 (487 bp)<br> 

&nbsp;[[Image:0613 Reamp Digestion.JPG]]<br> 

Unsuccessful<br> 

Date:&nbsp;June 14, 2012<br> 

Reamplified PCR&nbsp;product, see gel lanes 1-3<br> 

[[Image:0614 Amp.JPG]]<br> 

Lane 1: KTaq1-1 - KTaq1-16 (636 bp) 

Lane 2: KTaq2-1 - KTaq2-16 (629 bp) 

Lane 3: KTaq3-1 - KTaq3-14 (487 bp)<br> 

Successful amplifacation, cut out each band, purify each, join them together by Gisbon (~1.67 uL&nbsp;of each fragment, ran gel of joined template<br> 

[[Image:1151 0614 Amp.JPG]] 

Expected size (~1.9 kb) 

[[Image:1151 Digestion 0614.JPG]]<br> 

Subcloing ToughTaq from 1094 pET30b-ToughTaq to pET30b-sso7d-NK 369 using Kpn1 + Xho1<br> 

Digested ToughTaq and vector 369 with Kpn +&nbsp;Xho1, cut out bands, and purified DNA. 

Ligating over night 

Date:&nbsp;June 15, 2012<br> 

Transformed the ligated DNA&nbsp;from yesterday with DH5 alpha cells and plated on KAN plates<br> 

Also, redid the PCR&nbsp;amplication using PHUSION&nbsp;to join the fragments (1 min 30 sec extension time, 20 cycles)&nbsp;instead of Gibson<br> 

[[Image:1151 Amp 0615.JPG]]<br> 

Cut out band (~1.9 kb)&nbsp;and purified DNA, Digested ToughTaq and vector 369 with Kpn + Xho1<br> 

[[Image:1152 Amp Gibson.JPG]] 

Cut out the digestion (see lanes 3 and 4), purified and ligated for 2 hours <br>Transformed the ligated DNA with DH5 alpha cells and plated on KAN plates<br> 

Date: June 22, 2012 

[[Image:0622 Digestion.JPG]] 

Combined by Gibson, no band (expected:&nbsp;1.9kb) 

Date:&nbsp;June 25, 2012 

Ran gel for PCR&nbsp;product of fragments joined with PCR&nbsp;and ligase 

[[Image:1151 0625 amp.JPG]] 

No clear band where it is supposed to be (expected = 1.9 kb) 

=== Colony Screening  ===

[[Image:1151 Screening.JPG]] 

All negative, need to reclone 

[[Image:1197 Digestion 0619.JPG]] 

Digested 1151 with enzymes, incorrect band size (lanes 5 and 6) 

Date:&nbsp;June 21, 2012 

[[Image:1151 1151 Screening.JPG]] 

Digested 1151 with enzymes, did not run gel long enough. Redigest sample 5 (lane 5), may be the correct size? 

[[Image:0621 redigest.JPG]] 

Redigested samples 5 and 6, band is supposed to be 1.7 kb, may send sample 5 to sequencing 

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