Xiuli.huang at 18:32, 19 September 2012

2012-09-19T18:32:00Z

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

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

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

'''Notebook: '''

'''Page:'''

'''Clone ID: 1373'''

'''Experimenters: '''xiuli

== '''Results''' ==

=== Cloning ===

Date: 2012-5-29 

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

Gene synthesis by PCR, cut out band, purify and digest with Nde1 + Xho1, ligate with  Nde1 + Xho1 digested pDB.His.GST 433 

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

 

[[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 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 pDB.His.GST vector with Nde1 + Xho1

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

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 enzyme?

 

Redid amplication with KAPA 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 

[[Image:06062012 Digestion.JPG]]

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

Synthesized PCR product again, unsuccessful. 

[[Image:1197 0607 AMP.JPG]]

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

[[Image:1197 0607 amp 2.JPG]] 

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 

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) 

[[Image:1197 Digestion 0619.JPG]] 

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

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

Date: June 18, 2012

[[Image:0618 Digest.JPG]]

Reamplifed gene, see lans 5 an 6. Very blurry

 Date: June 19, 2012

Attempted to synthesize DNA again, a lot of smearing

[[Image:1197 0619.JPG]]

 

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

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

Date: June 20, 2012 

[[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 very blurry

Redid amplifaction: 

[[Image:1197 smear.JPG]]

Left: KAPA, Right: PHUSION

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

Date: June 21, 2012 

Reamplified 1197 with KAPA enzyme (40 seconds extension, 20 cycles and 0.5 uL of template) 

[[Image:1197 kapa amp.JPG]] 

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

[[Image:1197 digestion 0621.JPG]]

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

Purified DNA and ligated

Date: June 22, 2012 

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

[[Image:0622 Amp.JPG]] 

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

Date: June 25. 2012

[[Image:0625 Digestion.JPG]]

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

=== Colony Screening ===

[[Image:0608 PCR Screening.JPG]]

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

[[Image:1197 1155 Digestion.JPG]]

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

Date: 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) for sequencing

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

 

== '''Conclusion''' ==

 

[[Category:Cloning]]

1197 pDB.His.GST-3C - Revision history

Xiuli.huang at 18:32, 19 September 2012