Paul.xu at 19:47, 27 July 2012

2012-07-27T19:47:48Z

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== '''Cloning of pET30b-sso7d-ToughTaq''' ==

'''Purpose:''' Cloning of pET30b-sso7d-ToughTaq by gene synthesis 

'''Date: '''2012-06-13

'''Notebook: '''

'''Page:'''

'''Clone ID: '''1151

'''Experimenters: '''Jennifer Truong

== '''Results''' ==

=== Cloning ===

Date: June 13, 2012

Synthesized template in 3 fragments (KTaq1, KTaq1 and KTaq3), PCR with KAPA enzyme for 5 cycles with 15 seconds extension time, conbined them, added first (KTaq1-1) and last (KTaq3-14) oligos , PCR with KAPA 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 

Lane 1: KTaq1-1 - KTaq1-16 (636 bp) 

Lane 2: KTaq2-1 - KTaq2-16 (629 bp) 

Lane 3: KTaq3-1 - KTaq3-14 (487 bp) 

 [[Image:0613 Reamp Digestion.JPG]] 

Unsuccessful 

Date: June 14, 2012 

Reamplified PCR product, see gel lanes 1-3 

[[Image:0614 Amp.JPG]] 

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

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

Lane 3: KTaq3-1 - KTaq3-14 (487 bp) 

Successful amplifacation, cut out each band, purify each, join them together by Gisbon (~1.67 uL of each fragment, ran gel of joined template 

[[Image:1151 0614 Amp.JPG]]

Expected size (~1.9 kb)

[[Image:1151 Digestion 0614.JPG]] 

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

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

Ligating over night

Date: June 15, 2012 

Transformed the ligated DNA from yesterday with DH5 alpha cells and plated on KAN plates 

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

[[Image:1151 Amp 0615.JPG]] 

Cut out band (~1.9 kb) and purified DNA, Digested ToughTaq and vector 369 with Kpn + Xho1 

[[Image:1152 Amp Gibson.JPG]]

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

Date: June 22, 2012

[[Image:0622 Digestion.JPG]]

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

Date: June 25, 2012

Ran gel for PCR product of fragments joined with PCR 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: 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

 

== '''Result intepretation/Summary''' ==

== '''Conclusion''' ==

 

== '''Cloning of 1151 pET30b-sso7d-ToughTaq''' ==

'''Purpose:''' Cloning of 1151 pET30b-sso7d-ToughTaq

'''Date: '''2012-7-9---2012-7-12

'''Notebook:0'''

'''Page:'''

'''Clone ID:1151'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1151-3PCR 7-9-2012.JPG]] 

Gene synthesis 3 fragments, 

lane1: KTaq1-1+KTaq1-16; 

lane2: KTaq2-1+KTaq2-16; 

lane3: KTaq3-1+KTaq3-14; 

 

[[Image:1151 PCR 7-10-2012.JPG]] 

lane1:PCR using phusion

tempalte:PCR1+PCR2+PCR3

primer:KTaq1-1+KTaq3-14; 

 

[[Image:1151+1254 clone 7-10-2012.JPG]] 

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 281px; height: 66px;"
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|   lane 3 
|   lane 4 
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| PCR product 
| pET30b-sso7d-NK 369 
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| enzymes 
| colspan="2" |            Kpn1 + Xho1 
|}

 

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=== Colony Screening ===

Date: 2012-7-12

Make miniprep and digest with  Kpn1 + Xho1

[[Image:1151+1222 digest detect 7-12-2012.JPG]][[Image:1151_digest_detect_7-27-2012.JPG]] 

clone 1,2,3,4,5,6,7,8,9 may be right, need to sequence

 

== '''Result intepretation/Summary''' ==

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== '''Conclusion''' ==

&lt;br&gt;

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[[Category:Cloning]]

1151 pET30b-sso7d-ToughTaq - Revision history

Paul.xu at 19:47, 27 July 2012