1. preparation of vector: 1.1. vector: _________ 1.2. vector resistance: _________ 1.3. vector size: _______ kb 1.4. vector was: 1.4.1.

___ Double digested with ____ units or ___ ul of REN ___ and__ units or ___ ul of REN _____ in ___ul buffer. 1.4.2.___ Sequentially digested first with ____ units or ___ ul of REN ___ in __ ul volume, and then adding __units or __ ul of REN ____adding ___ul buffer ___. 1.4.2.1.vector was purified by ___ column; __ agarose gel; or __ precipitation. 1.4.3.___ dephosphorylated; ___ not dephosphorylated ; __ end filled. 1.4.3.1.vector was purified by ___ column; ___ agarose gel; __ precipitaton. 2. preparation of insert: 2.1. insert size: __ bp 2.2. insert was: 2.2.1.___ PCR amplified with primers including REN sites _____ and ___ by the polymerase (PCR enzyme) ____ in __ volume reaction. 2.2.1.1.Insert was purified after PCR by ___ column; ___ agarose. 2.2.2.___ PCR product treated to create blunt ends by enzyme ____; subsequently (digested by REN _____ to create one sticky end). 2.2.2.1.insert was purified by ___ column; ___ agarose; __ precipitation. 2.2.3.cut out of another vector by REN(s) ___ ( check here___ if sequential digest) in a total volume of ____ ul with __ units or __ ul of REN ____ and __ units or __ ul of REN ____. 2.2.3.1.insert was purified after digest by ___ column; ___ agarose; __ precipitation. 3. ligation set up: 3.1. ratio of vector : insert is ___ : ___ 3.2. concentration of vector : insert is ____ ng : ____ ng in __ volume. 3.3. ligation was done @ ___ ºC for ___ o/n; ___ hrs with T4 ligase. 4. transformation: 4.1. transformation is done by ___ calciumchloride/heat shock; ___ electroporation with __ ul bacteria strain ______. 4.2. transformation set up included following controls: 4.2.1.___ positive control; a known unaltered plasmid (expect a lot of colonies) 4.2.2.___ negative control; linearized vector submitted to ligation 4.2.2.1.___ dephosphorylated vector. (expect no colonies) 4.2.2.2.___ not dephosphorylated vector.

4.2.2.2.1.___ expect lots of colonies if both ends are blunt, same REN site or have same overhangs 4.2.2.2.2.___ expect no/few colonies if both ends have different REN sites 4.3. Bacteria were regenerated @ 37ºC in ____ul media 4.4. __ ul bacteria were streaked out on ____ plates 5. results: 5.1. ______ colonies for positive control 5.2. ______ colonies for negative control 5.3. ______ colonies for new/experimental cloning 5.3.1.__Clones were obtained but had following problems:____

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