1197 pDB.His.GST-3C
Cloning of 1197 pDB.His.GST-3C
[edit]Purpose: subcloning of 3C into pDB.His.MBP
Date: 2012-09-18
Notebook:
Page:
Clone ID: 1373
Experimenters: xiuli
Results
[edit]Cloning
[edit]Date: 2012-5-29
Gene synthesis by PCR, cut out band, purify and digest with Nde1 + Xho1, ligate with Nde1 + Xho1 digested pDB.His.GST 433
Vector (2) PCR product (1) digested with Nde1 and Xho1. Cut out, gel purify, elude with water. Date: May 31, 2012
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
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:
Cut out bands, purified, eluted with 20 uL water
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
No bands present, redo AGAIN, maybe it is the FHUSION enzyme?
Redid amplication with KAPA enzyme, cut out band for purification and digestion
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
06/07/2012: Again, no growth on plates.
Synthesized PCR product again, unsuccessful.
successful amplifacation of gene, cut out top band, purified DNA, digested DNA and vector for digestion
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
Date: June 14, 2012
Sequencing was all bad, restarted from the beginning. Amplifaction of template
Let gel run too long by accident, but going to cut out the bottom the gel for purifacation and digestion (see lane one)
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
Reamplifed gene, see lans 5 an 6. Very blurry
Date: June 19, 2012
Attempted to synthesize DNA again, a lot of smearing
|
Lane |
Amount of Template |
| 1 | 0.25 uL |
| 2 | 0.50 uL |
| 3 | 1.0 uL |
Date: June 20, 2012
Reamplifed 1197 (see lanes 8 and 9), purified and digested with Nde1 and Xho1
Digestion of DNA very blurry
Redid amplifaction:
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)
Successful amplifaction, cut out the top band for digestion with v48 with Nde1 and Xho1
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
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
Digested 1197 and vector 433 (v48) with Ndel and Xho1, cut out bands and purified, ligated
Colony Screening
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Only sample 2 positive (lane 9) grew in KAN media overnight
Did MiniPrep and digested plasmid to check for inserts, submitted sample 2 (lane 2) for sequencing\
Date: June 26, 2012
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
All clones positive (expected size = ~600 bp)
Submitted sample 2 (lane 2) for sequencing
Result intepretation/Summary
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Conclusion
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