Editing 1197 pDB.His.GST-3C

== '''Cloning of 1197&nbsp;pDB.His.GST-3C'''  ==

'''Purpose:''' subcloning of 3C into pDB.His.MBP<br>

'''Date: '''2012-09-18

'''Notebook:&nbsp;''' 

'''Page:''' 

'''Clone ID: 1373''' 

'''Experimenters: '''xiuli

== '''Results'''  ==

=== Cloning  ===

Date: 2012-5-29<br> 

[[Image:1197 gene synthesis 5 29 2012.JPG]]<br> 

Gene synthesis by PCR, cut out band, purify and digest with Nde1 + Xho1, ligate with&nbsp; Nde1 + Xho1 digested pDB.His.GST 433<br> 

[[Image:1197 Digestion 5 29 2012.jpg]]<br><br><br> Vector (2) PCR&nbsp;product (1) digested with Nde1 and Xho1. Cut out, gel purify, elude with water. Date: May 31, 2012<br> 

<br> 

[[Image:1197 PCR Screening 05312012 1.JPG]] 

Grew bacteria from lanes 5, 6, 8, 9, 11 and 12 

Date 6/1/2012: Bacteria did not grow in 5 of the tubes, only grew from Lane 8 

Digested sample 8 with&nbsp;Nde1 and Xho1 

[[Image:1197 Digest.JPG]] 

Submitted sample for sequencing 

Date: 06/04/2012 Sequencing came back bad (contaminated) picked up new colonies and grew them in media (7 samples) 

06/05/2012: Samples did not grow in the media, started experiment over again 

Made PCR product using PHUSION enzyme: 

[[Image:1197 PCR Product.JPG]] 

Cut out bands, purified, eluted with 20 uL water 

[[Image:1197 Digestion.JPG]] 

Digested DNA PCR product and&nbsp;pDB.His.GST vector&nbsp;with&nbsp;Nde1 + Xho1 

Cut out bands, purified, ligated, transformed with DH5 alpha cells, grow on KAN plates&nbsp; 

6/6/2012: No growth on plates, may have cut out incorrect band yesterday 

Redid gene synthesis today 

[[Image:1197 Amp.JPG]] 

No bands present, redo AGAIN, maybe it is the FHUSION&nbsp;enzyme? 

<br> 

Redid amplication with KAPA&nbsp;enzyme, cut out band for purification and digestion 

[[Image:1197 amp 2.JPG]] 

Digested DNA PCR product and pDB.His.GST vector with Nde1 + Xho1 

Cut out bands, purified, ligated, transformed with DH5 alpha cells, grow on KAN plates <br> 

[[Image:06062012 Digestion.JPG]] 

06/07/2012: Again, no growth on plates. 

Synthesized PCR product again, unsuccessful.<br> 

[[Image:1197 0607 AMP.JPG]] 

successful amplifacation of gene, cut out top band, purified DNA, digested DNA and vector for digestion<br> 

[[Image:1197 0607 amp 2.JPG]]<br> 

Digested DNA PCR product and pDB.His.GST vector with Nde1 + Xho1 

[[Image:1197 0607 Digestion.JPG]] 

Cut out bands, purified, ligated, transformed with DH5 alpha cells, grow on KAN plates <br> 

Date: June 14, 2012 

Sequencing was all bad, restarted from the beginning. Amplifaction of template 

[[Image:0614 amp 2.JPG]] 

Let gel run too long by accident, but going to cut out the bottom the gel for purifacation and digestion (see lane one)<br> 

[[Image:1197 Digestion 0619.JPG]]<br> 

Digested 1197 with enzymes, incorrect band size (lanes 1 - 4) 

Reamplified 1197 with PHUSION, a lot of smearing (see lane 7) <br> 

Date:&nbsp;June 18, 2012 

[[Image:0618 Digest.JPG]] 

Reamplifed gene, see lans 5 an 6. Very blurry 

<br> Date:&nbsp;June 19, 2012 

Attempted to synthesize DNA again, a lot of smearing 

[[Image:1197 0619.JPG]] 

<br> 

{| cellspacing="1" cellpadding="1" border="1" style="width: 200px; height: 88px;"
|-
| 
Lane 

| Amount of Template
|-
| 1 
| 0.25 uL&nbsp;
|-
| 2 
| 0.50 uL
|-
| 3 
| 1.0 uL
|}

Date:&nbsp;June 20, 2012<br> 

[[Image:1197 0620amp.JPG]] 

Reamplifed 1197 (see lanes 8 and 9), purified and digested with Nde1 and Xho1 

[[Image:1197 0620 Digestion.JPG]] 

Digestion of DNA&nbsp;very blurry 

Redid amplifaction:&nbsp; 

[[Image:1197 smear.JPG]] 

Left:&nbsp;KAPA, Right:&nbsp;PHUSION 

Still very blurry, used 0.5 uL&nbsp;of template NO&nbsp;CLEAR&nbsp;BAND 

Date:&nbsp;June 21, 2012<br> 

Reamplified 1197 with KAPA&nbsp;enzyme (40 seconds extension, 20 cycles and 0.5 uL&nbsp;of template)<br> 

[[Image:1197 kapa amp.JPG]]<br> 

Successful amplifaction, cut out the top band for digestion with v48 with Nde1 and Xho1<br> 

[[Image:1197 digestion 0621.JPG]] 

Succesful digestion, cut out circled bands in lane 7 and 8 

Purified DNA&nbsp;and ligated 

Date:&nbsp;June 22, 2012<br> 

Tranformed with Top 10 cells and plated on KAN&nbsp;resistant plates (to grow over the weekend) - Paul said sequencing for this may be bad because I&nbsp;used 0.5 uL&nbsp;of KAPA&nbsp;enzyme, reamplifed with only 0.25 uL&nbsp;of KAPA&nbsp;enzyme<br> 

[[Image:0622 Amp.JPG]]<br> 

Cut out band in lane 4 (expected size = 600 bp), placed in tube in gel, will purify and digest on Monday<br> 

Date: June 25. 2012 

[[Image:0625 Digestion.JPG]] 

Digested 1197 and vector 433 (v48)&nbsp;with Ndel and Xho1, cut out bands and purified, ligated 

=== Colony Screening  ===

[[Image:0608 PCR Screening.JPG]] 

Only sample 2 positive (lane 9)&nbsp;grew in&nbsp;KAN&nbsp;media overnight 

[[Image:1197 1155 Digestion.JPG]] 

Did MiniPrep and digested plasmid to check for inserts, submitted sample 2 (lane 2)&nbsp;for sequencing\ 

Date:&nbsp;June 26, 2012 

[[Image:0626 Screening.JPG]] 

All positive clones, grew samples 1-6 (lanes 1-6) in media over night 

Date: June 27, 2012 

Digested Miniprep with enzymes, check if insert is still present 

[[Image:0627 Screening.JPG]] 

All clones positive (expected size = ~600 bp) 

Submitted sample 2 (lane 2)&nbsp;for sequencing 

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

<br> 

== '''Conclusion'''  ==

<br> 

[[Category:Cloning]]